Post on 12-Oct-2020
Axiom™ 2.0 Assay 96-Array Format Automated WorkflowUSER GUIDE
for Biomek FXP (Windows® 7 and XP)
Publication Number 702963
Revision 5
For Research Use Only. Not for use in diagnostic procedures.
The information in this guide is subject to change without notice.
DISCLAIMER
TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Revision history
Important Licensing Information
This product may be covered by one or more Limited Use Label Licenses. By use of this product, you accept the terms and conditions of all applicable Limited Use Label Licenses.
Corporate entity
Life Technologies | Carlsbad, CA 92008 USA | Toll Free in USA 1 800 955 6288
TRADEMARKS
All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Jitterbug is a trademark of Boekel Scientific. Microsoft, Excel, and Windows are either registered trademarks or trademarks of Microsoft Corporation in the United States and/or other countries. QIAGEN and REPLI-g are registered or registration-pending trademarks of the QIAGEN Group. Beckman Coulter, and Biomek are either registered trademarks or trademarks of Beckman Coulter, Inc. TRobot is a trademark of Biometra GmbH. DNA Engine Tetrad, Bio-Rad, Microseal, and Hard-Shell are registered trademarks of Bio-Rad Laboratories, Inc.
©2018 Thermo Fisher Scientific Inc. All rights reserved.
Manufacturer:Affymetrix Pte Ltd 7 Gul Circle #2M-01Keppel Logistics Building Singapore 629563
Products:Axiom™ Array Plates
Manufacturer:Thermo Fisher Scientific Baltics UAB V.A. Graiciuno 8, LT-02241Vilnius, Lithuania
Products:Axiom™ 2.0 Reagent Kit
Table 1 Revision history of Pub. no. 702963
Revision Date Description
5 04 September 2018 Baseline for revision history.Updated to the current document template, with associated updates to trademarks, logos, licensing, and warranty.Updated to reflect that Axiom™ Reference gDNA 103 has been removed from the reagent kit and has been made available for purchase separately.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Contents
CHAPTER 1 The Axiom™ 2.0 Assay . . . . . . . . . . . . . . . . . . . . . . . . . . 10
About . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
What’s new . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Overview of the Axiom™ 2.0 Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Running multiple plate workflows . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
CHAPTER 2 Genomic DNA preparation and requirements. . . . . . . . . 13
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Sources of genomic DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
General requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Special requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Assessing the quality of genomic DNA using 1% Agarose E-gels . . . . . . . . . . . . . . . . . . 15
Genomic DNA extraction/purification methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Genomic DNA cleanup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Genomic DNA preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Equipment, consumables and reagents required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
1. Thaw samples and control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2. Quantitate and dilute gDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
3. Aliquot the diluted samples and the control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
4. Freeze or proceed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
5. Create a Batch Registration file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
CHAPTER 3 Target preparation on the Biomek FXP with Windows® XP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Axiom™ 2.0 Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Before using the Biomek workstation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Seal, vortex, and centrifuge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Breaking the light curtain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Pipette tip usage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Plate centrifuge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Set the Biomek software default settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3
Contents
Equipment, consumables, labware, and reagents required . . . . . . . . . . . . . . . . . . . . . . . . . 29
Labware and materials used on the Biomek™ workstation deck . . . . . . . . . . . . . . . . . . . 29
Proper tray alignment and loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Stain trays and covers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Loading tray consumables onto the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . 46
Reagent block template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Reservoir labels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Related Biomek FXP Target Prep Express documentation . . . . . . . . . . . . . . . . . . . . . . . . 50
Stage 1: DNA Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Equipment, consumables, labware, and reagents required . . . . . . . . . . . . . . . . . . . . . . . 51
1. Perform the pre-run checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
2. Thaw and prepare the reagents and Sample Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
3. Run the DNA Amplification step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Summary of DNA Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Stage 2: Fragmentation and Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Equipment, consumables, labware, and reagents required . . . . . . . . . . . . . . . . . . . . . . . 65
1. Perform the pre-run checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
2. Thaw and prepare the amplified DNA samples and reagents . . . . . . . . . . . . . . . . . . . . 67
3. Run the Fragmentation step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Summary of Fragmentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
4. Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
5. Centrifuge and dry pellets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Stage 3: Resuspension and hybridization preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Equipment, consumables, labware, and reagents required . . . . . . . . . . . . . . . . . . . . . . . 76
1. Preparing frozen pellets and Axiom Resusp Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
2. Perform the pre-run checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
3. Thaw and prepare the reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
4. Run the Resuspension and Hybridization Preparation step . . . . . . . . . . . . . . . . . . . . . 78
Summary of Resuspension and Hybridization Preparation . . . . . . . . . . . . . . . . . . . . . . . . 85
Stage 4: Preparation for the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
About Stage 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Duration of GeneTitan™ MC Instrument reagent preparation and sample preparation . . . 93
Equipment and consumables required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
1. Perform the pre-run checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
2. Prepare the reagents for GeneTitan Reagent Tray Preparation . . . . . . . . . . . . . . . . . . . 96
3. Prepare the Sample Plate (if stored at –20°C) and the array plate . . . . . . . . . . . . . . . . 98
4. Prepare the GeneTitan™ MC Instrument. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
5. Run the Preparation for GeneTitan™ step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
4 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Contents
6a. Complete Stage 4: Preparation for GeneTitan™ - Hybridization Trays . . . . . . . . . . . . 106
6b. Complete Stage 4: Preparation for GeneTitan™ - Reagent Trays. . . . . . . . . . . . . . . . 107
6c. Complete Stage 4: Preparation for GeneTitan™ - multiple plate workflow . . . . . . . . 107
Summary of Preparation for GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . 109
CHAPTER 4 Target preparation on the Biomek FXP with Windows® 7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
What's new for the Axiom™ 2.0 Target Preparation 96-Samples Biomek® FXP
Method for Windows® 7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
New features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
Method changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
New off-deck step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Axiom™ 2.0 Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Before using the Biomek workstation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Seal, vortex and centrifuge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Breaking the light curtain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
Pipette tip usage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
Plate centrifuge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Setting method preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Equipment, consumables, labware, and reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Labware and materials used on the Biomek workstation deck . . . . . . . . . . . . . . . . . . . . 124
Proper tray alignment and loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
Stain trays and covers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Loading tray consumables onto the GeneTitan™ MC Instrument. . . . . . . . . . . . . . . . . . . 140
Reagent block template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
Reservoir stickers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
Related Biomek FXP Target Prep Express documentation . . . . . . . . . . . . . . . . . . . . . . . 144
Stage 1: DNA Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Equipment, consumables, labware and reagents required . . . . . . . . . . . . . . . . . . . . . . . 145
1. Perform the pre-run checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
2. Thaw and prepare the reagents and Sample Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
3. Run the DNA Amplification step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
Summary of DNA Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
Stage 2: Fragmentation and Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Equipment, consumables, labware and reagents required . . . . . . . . . . . . . . . . . . . . . . . 157
1. Perform the pre-run checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
2. Thaw and prepare the amplified DNA samples and reagents . . . . . . . . . . . . . . . . . . . 159
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 5
Contents
3. Run the Fragmentation step . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
4. Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Summary of Fragmentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
Stage 3: Centrifugation and drying pellets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Equipment and consumables required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Stage 4: Resuspension, Hybridization Preparation, and QC . . . . . . . . . . . . . . . . . . . . . . . . 171
Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
Equipment, consumables, labware and reagents required . . . . . . . . . . . . . . . . . . . . . . . 171
1. Preparing frozen pellets and Axiom Resusp Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
2. Perform the pre-run checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
3. Thaw and prepare the reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
4. Run the Resuspension, Hybridization Preparation, and QC step . . . . . . . . . . . . . . . . 173
Summary of Resuspension and Hybridization Preparation . . . . . . . . . . . . . . . . . . . . . . . 180
Stage 5: Preparation for the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . 184
About Stage 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
Duration of GeneTitan™ MC Instrument reagent preparation and sample preparation . . 188
Equipment and consumables required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
1. Perform the pre-run checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
2. Prepare the reagents for GeneTitan Reagent Tray Preparation . . . . . . . . . . . . . . . . . . 191
3. Prepare the Sample Plate (if stored at –20°C) and the array plate . . . . . . . . . . . . . . . . 193
4. Prepare the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
5. Run the Preparation for GeneTitan™ step. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
6a. Complete Stage 4: Preparation for GeneTitan™ - Hybridization Trays . . . . . . . . . . . . 201
6b. Complete Stage 4: Preparation for GeneTitan™ - Reagent Trays . . . . . . . . . . . . . . . 202
6c. Complete Stage 4: Preparation for GeneTitan™ - Multiple Plate Workflow . . . . . . . . 202
Summary of Preparation for GeneTitan MC™ Instrument . . . . . . . . . . . . . . . . . . . . . . . . 204
CHAPTER 5 Array processing with the GeneTitan™ MC Instrument. 209
Before using the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
Proper tray alignment and loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
Stain trays and covers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
Email and telephone notifications from the GeneTitan™ MC Instrument . . . . . . . . . . . . . 214
GeneTitan™ MC Instrument lamp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
Setup options for array plate processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
Aborting a process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
Stage 1: Create and upload Batch Registration file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
Stage 2: Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
Reagents required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
Setup the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
6 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Contents
Load Axiom™ array plate and hybridization tray onto the GeneTitan™ MC Instrument . . 225
Load a second Axiom™ array plate and hybridization tray onto the
GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
Status window prompts and actions required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
Stage 3: Ligate, Wash, Stain, and Scan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
Equipment, consumables, and reagents required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
Proper installation of the GeneTitan™ tray consumables . . . . . . . . . . . . . . . . . . . . . . . . . 236
Load trays onto the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
Continuing the workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
Shutting down the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
CHAPTER 6 Processing 8 Axiom™ array plates per week. . . . . . . . . 246
Overview of the 8-plate workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
Plate numbering scheme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
Week 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
Week 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248
Thawing frozen plates of amplified DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
Automated target preparation for processing 8 Axiom™ array plates per week . . . . . . . . . 249
Initial target prep week—Biomek FXP Target Prep Express . . . . . . . . . . . . . . . . . . . . . . . . 250
Initial target prep week—Day 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
Initial target prep week—Day 2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
Initial target prep week—Day 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
Initial target prep week—Day 4. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254
Initial target prep week—Day 5. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
Simultaneous 8-plate workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
Eight-plate workflow—Day 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
Eight-plate workflow—Day 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259
Eight-plate workflow—Day 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262
Eight-plate workflow—Day 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
Eight-plate workflow—Day 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268
CHAPTER 7 Processing 3 Axiom™ array plates per week. . . . . . . . . 271
Overview of the 3-plate workflow for automated target preparation . . . . . . . . . . . . . . . . . . 272
Thawing frozen plates of amplified DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
Target prep and array processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
Three plate/week workflow—Day 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
Three plate/week workflow—Day 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276
Three plate/week workflow—Day 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
Three plate/week workflow—Day 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
Three plate/week workflow—Day 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 7
Contents
CHAPTER 8 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
Biomek FXP Target Prep Express . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
GeneTitan™ Multi-Channel Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
Miscellaneous messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282
Fluidic diagnostic messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284
Wash/Scan Resume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288
Aborting a run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288
APPENDIX A Safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
General safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
APPENDIX B Fragmentation quality control gel protocol . . . . . . . . . 292
Protocol for running a fragmentation quality control gel . . . . . . . . . . . . . . . . . . . . . . . . . . . 292
Equipment required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292
E-Gels and reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292
Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292
Diluting the TrackIt™ Cyan/Orange Loading Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
Fragmentation QC gel protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
APPENDIX C Sample quantitation after resuspension . . . . . . . . . . . 295
Protocol for sample quantitation after resuspension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
Equipment required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
Quantitate the diluted samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
Assess the OD readings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296
Suggested protocol for OD quantitation using the DTX 880 . . . . . . . . . . . . . . . . . . . . . . . . 297
If performing sample quantitation on a plate reader other than the DTX880 . . . . . . . . . . . 303
APPENDIX D Registering samples in GeneChip™ Command Console™ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304
Creating a GeneTitan™ Array Plate Registration file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304
8 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Contents
APPENDIX E Deionization procedure for GeneTitan™ trays and covers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
Deionization procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
Ion-indicator cap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310
APPENDIX F GeneTitan™ Multi-Channel Instrument Care . . . . . . . . 311
Cleaning and maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
Monthly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
Every 6 months . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
Servicing the outer enclosure fan filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
Cleaning schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
Cleaning procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
Replacing the bottle filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313
Removing and inspecting the filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314
Replacing the filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314
Replacing the xenon lamp in the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . . 315
Lamp life/imaging device status notices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
Removing the xenon lamp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316
Replacing the lamp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317
Resetting the lamp counter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320
Log files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320
GCC log files for GeneTitan™ MC Instrument Systems . . . . . . . . . . . . . . . . . . . . . . . . . . 321
Insufficient disk space notice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322
Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323
Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323
Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 9
1 The Axiom™ 2.0 Assay
About
The first genome-wide association study (GWAS) was published in 2005 (Manolio and Collins) when individuals carrying particular haplotypes of SNP rs380390 were found to have increased risk of developing age-related macular degeneration, a study performed with the Applied Biosystems GeneChip Mapping 100K Array Set (Klein et al.).
As of September, 2009, there have been over 400 peer-reviewed GWAS publications and over 1774 SNPs have been implicated in human disease (Hindorff et al.). Initial GWAS studies focused on the “common disease, common variant” hypothesis (Manolio and Collins) that held that haplotypes with a minor allele frequency (MAF) 5% would show measurable contribution to human disease research.
Current research is shifting towards “complex disease, complex/rare variant” studies. As such, these research projects require a broader catalog of human variation, such as is being generated by the 1000 Genomes Project (http://www.1000genomes.org). This project focuses on identifying alleles with a MAF 5% across a broader spectrum of human ethnicities. In order to allow our customers to take advantage of this novel and rare content for genome association and candidate gene studies in a cost effective and timely manner, Thermo Fisher Scientific is introducing a new genotyping product line: the Axiom™ Genotyping Solution.
The Axiom Genotyping Solution introduces a new genotyping technology platform that includes novel assay biochemistry, array configuration and processing, and automated target preparation. This solution has applications in human disease research and basic and applied agriculture research.
For human disease research applications, Thermo Fisher Scientific conducted an empirical screen of genomic content from dbSNP (http://www.ncbi.nlm.nih.gove/projects/SNP/). The screen included markers from HapMap and the 1000 Genomes Project as well as other sources, using HapMap phase 3 samples and/or the original 270 HapMap samples. All of this information has gone into creating a proprietary Thermo Fisher Scientific database of validated markers that can be interrogated using the Axiom™ 2.0 Assay.
There are several arrays available for use with the Axiom 2.0 Assay which leverage the content of this proprietary Thermo Fisher Scientific database. For a complete list of supporting products visit www.thermofisher.com.
10 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 1 The Axiom™ 2.0 AssayWhat’s new 1
The Axiom 2.0 Assay interrogates biallelic SNPs and simple indels (human only) in a single, fully automated assay workflow. Starting with genomic DNA, the samples are processed by performing either an automatic or manual target preparation protocol followed by automated processing of the array plates in the GeneTitan MC Instrument.
• Target preparation uses methods including DNA amplification, fragmentation, purification and resuspension of the target in hybridization cocktail.
• The hybridization-ready targets are then transferred to the GeneTitan™ Multi-Channel (MC) Instrument for automated, hands-free processing including hybridization, staining, washing and imaging.
Cel files generated by the GeneTitan MC Instrument are processed using the Axiom™ Genotyping Algorithm version 1 (Axiom GT1) available through Applied Biosystems™ Microarray Power Tools or Genotyping Console™ v4.1.
In summary, the Axiom Genotyping Solution is a product line that provides catalog arrays that:
• Are optimized for high genetic coverage of their population in question.
• Provide highly automated, reproducible results suitable for GWAS.
What’s new
In this version of the Axiom™ 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP User Guide instructions are provided for running the Biomek FXP Target Prep Instrument using either Windows® XP or Windows 7 operating system. Instructions for running Windows XP are provided in Chapter 3 and instructions for running Windows 7 are provided in Chapter 4.
Overview of the Axiom™ 2.0 Assay
Running the Axiom 2.0 Assay requires the following sets of steps:
1. Genomic DNA preparation—Resulting in samples that meet requirements spelled out in Chapter 2, ʺGenomic DNA preparation and requirementsʺ on page 13.
2. Target Preparation of the samples:
• Chapter 3, ʺTarget preparation on the Biomek FXP with Windows® XPʺ on page 22, or
• Chapter 4, ʺTarget preparation on the Biomek FXP with Windows® 7ʺ on page 114
3. Array processing, done with:
• GeneTitan MC Instrument
• GeneTitan Instrument Control software
• Applied Biosystems™ GeneChip™ Command Console Portal software
See Chapter 5, ʺArray processing with the GeneTitan™ MC Instrumentʺ on page 209.
A list of the required equipment and supplies for running the Axiom 2.0 Assay using the Biomek FXP for automated target preparation can be found in the Axiom™ 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP Site Preparation Guide, Pub. No. 702984.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 11
Chapter 1 The Axiom™ 2.0 AssayOverview of the Axiom™ 2.0 Assay1
Running multiple plate workflows
Thermo Fisher Scientific provides workflows that allow you to run a set of samples and array plates through the protocol using a minimum of personnel and a forty-hour week. The timing of steps is critical because of the following constraints:
• Incubation after DNA Amplification is 23 hours.
• Hybridization in the GeneTitan MC Instrument is 23.5 hours.
• Reagent trays for wash/stain/imaging must be prepared as Hybridization finishes.
• Limits to when a second hybridization tray and array plate can be loaded into the GeneTitan MC Instrument.
These limitations require careful timing. The details are covered in:
• Chapter 6, ʺProcessing 8 Axiom™ array plates per weekʺ on page 246.
• Chapter 7, ʺProcessing 3 Axiom™ array plates per weekʺ on page 271.
12 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
2 Genomic DNA preparation andrequirements
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Sources of genomic DNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
General requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Genomic DNA extraction/purification methods . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Genomic DNA cleanup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Genomic DNA preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Overview
The general requirements for genomic DNA (gDNA) sources and extraction methods are described in this chapter. The success of this assay requires uniform amplification of the genome starting with relatively intact gDNA. To achieve this, the gDNA must be of high quality, and must be free of contaminants that may affect the enzymatic reactions to be performed.
For this protocol, you use the Axiom™ 2.0 Reagent Kit (96 reaction, Cat. No. 901758). Axiom Reference Genomic DNA 103 (Cat. No. 951957) meets the requirements outlined below, and is used as a control. The size and purity of sample gDNA can be compared with those of the control DNA to assess sample quality. The control DNA should also be used routinely as an experimental positive control and for troubleshooting purposes.
Assay performance may vary for gDNA samples that do not meet the general requirements described below. However, the reliability of any given result should be assessed in the context of overall experimental design and goals.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 13
Chapter 2 Genomic DNA preparation and requirementsSources of genomic DNA2
Sources of genomic DNA
The following sources of human gDNA have been successfully tested in the laboratories at Thermo Fisher Scientific and meet the requirements outlined under ʺGeneral requirementsʺ on page 15.
• Blood
• Saliva
• Cell line
• WGA pre-amplified DNA: Genomic DNA amplified with the REPLI-g® Kit (a whole genome amplification kit; QIAGEN, Cat. No. 150025) has been tested successfully with the Axiom 2.0 Genome-Wide Human Reagent Kit Assay. The REPLI-g Kit was used to amplify 20 ng genomic DNA, and the resulting yields were quantitated by a PicoGreen® assay. The amplified products (either 100 or 200 ng amplified DNA as required according to the Axiom array type) were used (without purification) as the input DNA sample in the subsequent Axiom 2.0 Assay steps. The stability of this amplified product to storage and repeated cycles of freeze/thaw have not been evaluated by Thermo Fisher Scientific.
The following sources of animal gDNA have been successfully tested in the laboratories at Thermo Fisher Scientific and meet the requirements outlined under ʺGeneral requirementsʺ on page 15:
• Blood
• Semen
• Nasal swab
• Hair bulbs
• Ear punch tissue
The following sources of plant gDNA have been successfully tested in the laboratories at Thermo Fisher Scientific and meet the above requirements:
• Seeds
• Leaves
Note: DNA derived from formalin-fixed paraffin-embedded (FFPE) blocks should not be used with this assay.
Success with other types of samples will depend on quality (degree of degradation, level of purity, etc.) and quantity of gDNA extracted.
14 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 2 Genomic DNA preparation and requirementsGeneral requirements 2
General requirements
• Starting DNA must be double-stranded for the purpose of accurate concentration determination.
• DNA must be of high purity.DNA should be free of DNA polymerase inhibitors. Examples of inhibitors include high concentrations of heme (from blood) and high concentrations of chelating agents (i.e., EDTA). The gDNA extraction/ purification method should render DNA that is generally salt-free because high concentrations of particular salts can also inhibit enzyme reactions. DNA purity is indicated by OD260/OD280
and OD260/OD230 ratios. The OD260/OD280 ratio should be between 1.8 and 2.0 and the OD260/OD230 ratio should be greater than 1.5. We recommend that DNA samples that do not meet these criteria be cleaned up as described under ʺGenomic DNA cleanupʺ on page 17.
• DNA must not be degraded.The approximate average size of gDNA may be assessed on a 1% agarose gel using an appropriate size standard control. Approximately 90% of the DNA must be greater than 10 Kb in size. Control DNA can be run on the same gel for side-by-side comparison.
Special requirements
Preamplification areaPrecautions are required when manipulating genomic DNA to avoid contamination with foreign DNA amplified in other reactions and procedures. It is recommended that genomic DNA manipulations are performed in a dedicated preamplification room or area separate from the main laboratory.
This preamplification area should have a dedicated set of pipettes and plasticware. If no dedicated area is available, use of a dedicated bench or a dedicated biosafety hood and dedicated pipettes is suggested. If no dedicated bench or biosafety hood is available, a set of dedicated pipettes is recommended.
Assessing the quality of genomic DNA using 1% Agarose E-gels
We recommend this quality control step to assess the quality of the gDNA prior to starting the assay.
Equipment and reagents recommended
Table 1 E-Gel® and reagents required
Item Cat. No
Mother E-Base Device EB-M03
Daughter E-Base Device EB-D03
E-Gel® 48 1% agarose gels G8008-01
RediLoad™ 750026
E-Gel® 96 High Range DNA Marker 12352-019
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 15
Chapter 2 Genomic DNA preparation and requirementsGeneral requirements2
Guidelines for preparing the Genomic DNA Plate for gel analysis• Loading a DNA mass of 10 ng to 20 ng per well is recommended. If lower amounts
are loaded, omission of the loading dye is recommended in order to improve visualization. Loading 25 ng gDNA per well can improve the image.
• Add 3 µL of 0.1X of RediLoad dye to each sample.
• Bring each sample to a total volume of 20 µL using H2O (for example, if the volume of genomic DNA is 5 µL, add 3 µL of RediLoad, and bring to 20 µL total by adding 12 µL of H2O).
• Seal, vortex and centrifuge.
Run a 48-lane 1% agarose e-gel
1. Power on for E-Base (red light).
2. Push the Power/Prg button to ensure that the program is at EG mode (not EP).
3. Insert the two 48-well 1% agarose e-gels into the slots.
4. Remove 2 combs.
5. Load 20 µL from the above plate onto two 48-well 1% agarose e-gels.
6. Load 15 µL of diluted High Range DNA Marker (1:3 dilution or ~0.34 X from stock) into all marker wells (as needed).
7. Fill all empty wells with water.
8. Adjust the run time to ~27 minutes.
9. Push the Power/Prg button again (it will change from red to green).
When run time is reached (the ladder band reaches the end of the lane), the system will automatically shut off. The gel is then ready for imaging.
Figure 1 shows gel images of intact gDNA (that is suitable for use in the Axiom™ 2.0 Assay) and degraded gDNA samples. Customers whose gDNA is degraded (similar to the image in Figure 1) should perform a test experiment to investigate the performance of their samples in the Axiom Genotyping Assay prior to beginning any large scale genotyping projects.
Figure 1 Gel images showing intact gDNA and degraded gDNA.
16 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 2 Genomic DNA preparation and requirementsGenomic DNA extraction/purification methods 2
Genomic DNA extraction/purification methods
Genomic DNA extraction and purification methods that meet the general requirements outlined above should yield successful results. Methods that include boiling or strong denaturants are not acceptable because the DNA would be rendered single-stranded and can no longer be accurately quantitated using a PicoGreen-based assay.
Genomic DNA cleanup
If a gDNA preparation is suspected to contain inhibitors, the following cleanup procedure can be used:
1. Add 0.5 volumes of 7.5 M NH4OAc, 2.5 volumes of absolute ethanol (stored at –20°C), to gDNA.
2. Vortex and incubate at –20°C for 1 hour.
3. Centrifuge at 12,000 x g in a microcentrifuge at room temperature for 20 minutes.
4. Remove supernatant and wash pellet with 80% ethanol.
5. Centrifuge at 12,000 x g at room temperature for 5 minutes.
6. Remove the 80% ethanol and repeat the 80% ethanol wash 1 more time.
7. Resuspend the pellet in reduced EDTA TE Buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA).
(See the Axiom™ Assay 96-Array Format Automated Workflow for Biomek FXP Site Preparation Guide, Pub. No. 702984 for reagents, equipment, labware and consumables for Axiom 2.0 Assay).
Genomic DNA preparation
This step needs to be done before proceeding with the DNA amplification stages for automated target preparation.
The genomic DNA (gDNA) you will process using the Axiom 2.0 Assay should meet the general requirements listed earlier in this chapter. The amount of gDNA depends on which Axiom array will be used in the downstream protocol. All human Axiom arrays (except the Axiom™ Genome-Wide Pan-African Array Set) require a total of 100 ng. The Axiom Genome-Wide Pan-African Array Set requires a total of 300 ng, or 100 ng per array (there are 3 arrays in the Axiom Genome-Wide Pan-African Array Set). Diploid plants and animals require 150 ng per array and polyploid plants and animals require 200 ng per array.
Table 2 Input requirements for Axiom™ 2.0 Assay
Sample type Volume per well
Input mass per well
gDNA concentration
Human 20 μL 100 ng 5 ng/μL
Diploid Plants and Animals 20 μL 150 ng 7.5 ng/μL
Polyploid Plants and Animals 20 μL 200 ng 10 ng/μL
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 17
Chapter 2 Genomic DNA preparation and requirementsGenomic DNA preparation2
To prepare gDNA
ʺ1. Thaw samples and controlʺ
ʺ2. Quantitate and dilute gDNAʺ.
ʺ3. Aliquot the diluted samples and the controlʺ
ʺ4. Freeze or proceedʺ
ʺ5. Create a Batch Registration fileʺ
Duration Thirty minutes to an hour for reagents to thaw and half an hour for setup.
Equipment, consumables and reagents required
Equipment and consumablesThe equipment and consumables listed in Table 3 are required for this stage.
Table 3 Equipment and consumables required for genomic DNA preparation
Quantity Item
As required Adhesive seals for plates
1 Ice bucket, filled with ice
1 each Pipettes:• Single channel P10 or P20 • Optional: multichannel P10 or P20
As required Pipette tips
1 Plate, deep well: Beckman Deep Well Titer, polypropylene; Cat. No. 2670071
1 Note that a different deep-well plate is used for each manual (96, 24, and Mini 96) and automated(NIMBUS 96 and Biomek FXP 384HT) workflow versions of the Axiom 2.0 Assay. See Chapter 2 of thespecific Axiom™ 2.0 Assay user guide for each workflow for further details.
1 Plate centrifuge
1 Plate spectrophotometer (required only if no OD measurements available for samples)
1 Vortexer
18 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 2 Genomic DNA preparation and requirementsGenomic DNA preparation 2
ReagentsThe reagents listed in Table 4 are required for this stage.
1. Thaw samples and control
Thaw the components listed below to room temperature:
• gDNA samples
• Axiom Reference Genomic DNA 103
To thaw, either:
• Place items on benchtop for 1 hour
• Thaw in a water bath:
a. Fill a small plastic dish with Millipore water. Do not overfill as the level of the water should not overflow when the sample tubes or plates are placed in the bath.
b. Thaw the sealed Sample Plate and reference sample for 30 minutes.
c. Remove the Sample Plate and/or sample tube from the water bath and wipe dry using laboratory tissues. Ensure the outside is completely dry before opening the Sample Plate or tube to minimize any contamination, which can lead to reaction failure.
2. Quantitate and dilute gDNA
1. Gently vortex (50% maximum) and centrifuge the gDNA and Reference Genomic DNA 103.
2. Recommendation: quantitate each sample (e.g., using the Quant-iT™ PicoGreen® dsDNA Kit).
3. Using reduced EDTA TE buffer, dilute each sample to a concentration of:
• 5 ng/µL for Human DNA samples
• 7.5 ng/µL for diploid plant and animal DNA samples
• 10 ng/µL for polyploid plant and animal DNA samples
4. Seal, vortex and centrifuge.
Note: Do not dilute the Reference Genomic DNA 103 control. It is already at a working concentration.
Table 4 Reagents required for genomic DNA preparation
Reagent Cat. No.
Axiom Reference Genomic DNA 103, –20°C(use as a positive control if genotyping human samples)
951957
Reduced EDTA TE Buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) 75793
Positive control gDNA (if genotyping non-human samples)
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 19
Chapter 2 Genomic DNA preparation and requirementsGenomic DNA preparation2
3. Aliquot the diluted samples and the control
Next, the samples and control are placed in the following deep well plate for target preparation:
• For automated target preparation: Beckman Deep Well Titer, polypropylene; Cat. No. 267007
1. 20 µL of each diluted gDNA sample (this should be the equivalent of 100 ng to 200 ng of gDNA, as required by the sample type).
2. 20 µL of control DNA.
We recommend including at least 1 positive control on each plate.
3. Seal and centrifuge.
Note: For samples to be processed on the Axiom Genome-Wide Pan-African Array Set, 3 identical deep well plates of 100 ng gDNA per well should be made.
4. Freeze or proceed
At this point you can:
• Store the Sample Plate at –20°C, or
• Proceed to DNA amplification (see Chapter 3, ̋ Target preparation on the Biomek FXP with Windows® XPʺ on page 22).
Note: You can leave the gDNA Sample Plate at room temperature if proceeding immediately to DNA Amplification.
5. Create a Batch Registration file
GeneTitan Array Plate Registration files contain information that is critical for:
• Data file generation during imaging.
• Tracking the experimental results for each sample loaded onto an array plate.
Detailed instructions for creating this file are located in Appendix D, ʺRegistering samples in GeneChip™ Command Console™ʺ on page 304. See also Figure 2 for a screen shot showing an example of a batch registration file.
1. Open GCC Portal Samples, and select:
a. GeneTitan Array Plate Registration.
b. The array plate format.
c. Click Download.
2. Enter a unique name for each sample and any additional information.
3. Save the file.
The array plate barcode will not be scanned until you are ready to load the array plate and samples onto the GeneTitan MC Instrument for processing.
IMPORTANT! It is very important to create and upload a GeneTitan™ Array Plate Registration file with your sample information prior to loading the array plate and hybridization tray into the GeneTitan MC Instrument. We recommend that you create (but not upload) this file at the same time you prepare your plate of genomic DNA. When your samples are ready for hybridization, you will scan the array plate barcode and upload the file to Applied Biosystems™ GeneChip™ Command Console (GCC).
20 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 2 Genomic DNA preparation and requirementsGenomic DNA preparation 2
Figure 2 Example of a Batch Registration file
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 21
3 Target preparation on the BiomekFXP with Windows® XP
This chapter is intended for users running the Axiom 2.0 automated target preparation method using the Biomek FXP Target Prep Express software on Windows XP. If you are currently using Windows® 7, see Chapter 4, ̋ Target preparation on the Biomek FXP with Windows® 7ʺ on page 114.
Axiom™ 2.0 Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Before using the Biomek workstation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Equipment, consumables, labware, and reagents required . . . . . . . . . . . . . . . . . 29
Stage 1: DNA Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Stage 2: Fragmentation and Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Stage 3: Resuspension and hybridization preparation . . . . . . . . . . . . . . . . . . . . . 76
Stage 4: Preparation for the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . 89
Axiom™ 2.0 Assay
The Axiom 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP instrument with Windows XP is designed for processing 96 samples at a time. The protocol is performed in 2 parts:
• Part 1: Target Preparation is performed on the Biomek FXP Target Prep Express
• Part 2: Array Processing is performed on the GeneTitan™ Multi-Channel (MC) Instrument
A list of all equipment and resources for the Axiom 2.0 Assay with Automated Target Preparation is in the Axiom™ 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP Site Preparation Guide, Pub. No. 702984.
This chapter includes instructions for Part 1: Target Preparation.
IMPORTANT! Before proceeding to DNA Amplification, do the gDNA preparation described in Chapter 2, ʺGenomic DNA preparation and requirementsʺ on page 13.
22 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPBefore using the Biomek workstation 3
Before using the Biomek workstation
Seal, vortex, and centrifuge
Unless otherwise stated in the protocol, follow the guidelines below when instructed to seal, vortex and centrifuge plates or reagent tubes for the Biomek FXP Target Prep Express portion of this assay.
• Seal plates—we recommend using MicroAmp™ Clear Adhesive Films to seal your plates.
• Vortex:
– Reagents 3 times, 1 second each time at the maximum setting.
– Plates 1 second each corner, and 1 second in the center at the maximum setting.
• Centrifuge: When instructed to centrifuge plates or reagent vials, follow these guidelines unless otherwise instructed (for example, when centrifuging and drying pellets, ʺ5. Centrifuge and dry pelletsʺ on page 75).
– Plates:
• Centrifuge at 1,000 rpm for 1 minute at room temperature.
• Do not centrifuge for more than 1 minute.
– Reagent Vials: 3 seconds
IMPORTANT! Always ensure that your plates are tightly sealed. A tight seal will prevent sample loss and cross-well contamination, particularly when plates are being vortexed. Never reuse a seal. always use a new seal.
Figure 3 Vortex plates at the corners and center
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 23
Chapter 3 Target preparation on the Biomek FXP with Windows® XPBefore using the Biomek workstation3
Breaking the light curtain
For your safety, the Biomek FXP Target Prep Express is designed to immediately halt all movement when the light curtain is broken.
Light curtain broken while running the assayFor your safety, the Biomek FXP Target Prep Express is equipped with a light curtain (Figure 4). The light curtain senses when an object (such as a hand or an arm) enters the space surrounding the deck. When this curtain is broken, all movement on the deck halts until the user either clicks OK to resume the activity that was taking place, or aborts the activity. Incubation timers are not interrupted.
Pipette tip usage
Figure 4 Prompt displayed when the light curtain is broken during the process referred to as Homing All Axes.
The light curtain covers the front of the instrument. The sides are protected by Plexiglas. Both areas are shown as red in this figure. Any penetration of the light curtain from outside the deck halts all movement of the workstation. To resume activity on the deck, click OK. To abort the step or other activity, click Stop on the toolbar.
Table 5 Pipette tip usage for 1 full run of the Axiom™ 2.0 Assay on the Biomek workstation
StepMultichannel
P50, pink96 tips, Cat. No. A21586
Multichannel AP96 P250,
aqua96 tips,
Cat. No. 717253
Span-8 Span P250,
green96 tips,
Cat. No. 379503
Span-8 Span P1000,
yellow96 tips,
Cat. No. 987925
96 rxns 96 rxns 96 rxns 96 rxns
DNA Amplification — 96 tips — 33 tips
Fragmentation — 96 tips 24 tips 20 tips
Resuspension and Hybridization Preparation 96 tips 96 tips 17 tips 23 tips
Preparation for the GeneTitan™ MC Instrument • Denature samples• Transfer denatured samples to hyb tray• Prepare GeneTitan™ reagent trays
— 96 tips 26 tips 96 tips
Total 96 tips 384 tips 67 tips 172 tips
24 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPBefore using the Biomek workstation 3
Plate centrifuge One plate centrifuge is required for the Axiom™ 2.0 Assay. See the Assay 96-Array Format Automated Workflow for Biomek FXP Site Preparation Guide, Pub. No. 702984, for an appropriate plate centrifuge that can be used with the Axiom Genotyping Solution. When centrifuging and drying pellets as instructed under ʺ5. Centrifuge and dry pelletsʺ on page 75, the centrifuge must be able to spin down plates at:
• rcf: 3,200 x g (4,000 rpm for the Eppendorf 5810R with the rotor configuration described in the Axiom™ 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP Site Preparation Guide)
• temperature: 4°C
In addition, the bottom of the rotor buckets should be soft rubber to ensure that the deep well plates do not crack. Do not centrifuge plates in metal or hard plastic buckets.
Set the Biomek software default settings
Typically you will select the default settings for the Biomek software once. The settings you select will:
• Determine the default step selected when the user prompt is displayed at the start of each run.
• Determine which process controls will be run during stages 2 and 3 (Fragmentation and Resuspension). The process controls include:
– Highly recommended: Preparation of sample dilution plates for OD and gel analysis during resuspension and hybridization preparation. The dilution plates are taken off-deck. One is used for OD quantitation to evaluate DNA mass; the other is used to check fragment size.
– If desired: Prompt you to manually take samples for DNA quantitation prior to fragmentation.
• Select deck configuration options for the type of integrated thermal cycler used.
To select Biomek software default settings:
1. Launch the Biomek Software.
2. Open Project Open Project Axiom 2.0 Target Prep and click OK (Figure 5).
3. Open File Open to display the Open Method window (Figure 6).
Or click the Open Method icon
4. Select Axiom 2.0 Target Prep and click OK.
Figure 5 Opening the Axiom 2.0 target preparation project
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 25
Chapter 3 Target preparation on the Biomek FXP with Windows® XPBefore using the Biomek workstation3
.
5. In the left pane of the window, select Axiom Target Prep (Figure 7).
The default settings window is displayed on the right.
Figure 6 Opening the Axiom target preparation method
The Axiom 2.0 Target Prep project folder must be displayed in this menu.
Figure 7 Click Startup Dialog to open the Default Settings window
The selections made in this box, Choose default settings, are displayed each time this window appears.
The options in the Select preferences box must be selected prior to starting a run.
You are not prompted to select any preferences at start up.
26 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPBefore using the Biomek workstation 3
6. Select your default settings.
Choose defaults settings—these settings can be changed at the start of each step
• Which step do you want to run?
The step that you choose will be the default setting for the runtime prompt. The actual step selected when the runtime prompt is shown reflects the state of the last completed run of the Axiom target preparation method. For example, if DNA Amplification was the step completed in the last run, then the step chosen in the subsequent run would be Fragmentation (the next step in the process). If there is no record of the previous run, then the default choice specified is selected.
Select preferences—These settings are displayed in this window only. You must select/deselect here.
• QC check points
• Prompt for manual DNA quantitation before fragmentation—the workstation will pause following inactivation of the DNA amplification reaction to allow you to manually remove an aliquot of each sample for off-line (manual) DNA quantitation. This extra quality control step is available for troubleshooting the DNA amplification reaction.
• Prepare plates for gel QC and OD after resuspension—the workstation will prepare 2 plates of resuspended samples properly diluted for the fragmentation gel QC and OD quantitation process control checks. See Appendix B, ̋ Fragmentation quality control gel protocolʺ on page 292 and Appendix C, ʺSample quantitation after resuspensionʺ on page 295 for instructions and result assessment guidelines.
• Custom run options
• Run method in test mode—select this option to skip all of the incubation timers in a step. If selected, a prompt is displayed asking you to confirm that you want to run a step in test mode (Figure 8). Use this option to perform runs using deionized water only, not actual reagents or samples.
IMPORTANT! For troubleshooting and support purposes, we strongly recommend that you perform the gel QC and OD quantitation process controls after resuspension.
CAUTION! Never process samples in test mode. The assay will fail; all of your samples and reagents will be lost.
Figure 8 Prompt displayed when the custom run option, Run method in test mode, is selected.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 27
Chapter 3 Target preparation on the Biomek FXP with Windows® XPBefore using the Biomek workstation3
• Deck configuration options
• TRobot—select this option to perform the denaturation of the Axiom Hyb Ready Plate on the integrated Biometra TRobot 96 thermal cycler
• PTC—select this option to perform the denaturation of the Axiom Hyb Ready Plate on the integrated Bio-Rad PTC-200 thermal cycler
• No integrated thermal cycler—select this option to perform the denaturation of the Hyb Ready Plate on an off-deck thermal cycler or if your Biomek does not have an integrated thermal cycler. A list of thermal cyclers that have been verified with the assay can be found below. When selecting this option, select the appropriate plate type that should be used for the Hyb Ready Plate.
– Select the Bio-Rad Skirted option when using the HSP-9631 plate for the PTC-200 or the Bio-Rad Tetrad 0240G thermal cycler.
– Select the Bio-Rad Semi-Skirted on Costar Round U-Bottom option for the Applied Biosystems 9700 or the Applied Biosystems 2720 thermal cycler. The Bio-Rad HSS-9601 plate must be stacked onto the Costar Round Bottom plate from Corning (VWR International Cat. No. 29442-392, E&K Scientific: EK 680568, Corning Mfg. Cat. No. 3795) for the Biomek FXP Target Prep Express to prepare the Hyb Ready Plate.
• Controllers—select the appropriate Peltier controller used by the Biomek FXP Target Prep Express.
– Black TEC Control
– Blue Single TEC Control
We have verified the performance of this assay using the Bio-Rad PTC-200/PTC-200G and Biometra TRobot 96 on the Beckman Biomek Target Prep Express liquid handler. We have also verified the performance of this assay using the following off-deck thermal cyclers, with 96-well blocks:
• Bio-Rad PTC-200G
• Biometra TRobot 96
• Applied Biosystems 9700 with a gold, silver or aluminum block
• Applied Biosystems 2720
• Bio-Rad / MJ Tetrad® 2 PTC-0240G
The performance of this assay has not been verified with other thermal cyclers.
Use of other thermal cyclers may result in assay failure and may violate the Axiom Array and Reagent replacement policy.
The thermocycler needs to be programmed with the Axiom Denature protocol:
a. 95°C 10 minutes
b. 48°C 3 minutes
c. 48°C hold
Use the heated lid option when setting up or running the protocol.
WARNING! Evaporation during denaturation can negatively impact assay performance. Use the recommended thermal cycler consumables and sealing film to eliminate condensation and evaporation. The arched, auto-sealing metal plate with P pads as shown in Table 6 on page 29 should be replaced as per the manufacturer’s recommendation.
28 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPEquipment, consumables, labware, and reagents required 3
7. Click Start at the top of the left pane to close the default settings window.
Equipment, consumables, labware, and reagents required
Labware and materials used on the Biomek™ workstation deck
IMPORTANT! The default settings you select here will be displayed each time the Axiom target preparation window is displayed.
The options displayed in the “Select preferences” section of the Startup dialog box as shown in Figure 7 on page 26 must be selected prior to starting a run. These settings are not prompted for at runtime.
Table 6 Labware and materials used on the Biomek™ workstation deck
Labware Supplier and Cat. No.
Labware image
Biomek AP96 – P250 Pipette Tips (aqua box; pre-sterile, barrier)
Beckman CoulterCat. No. 717253
Biomek Span P250 Pipette Tips (green box; pre-sterile, barrier)
Beckman CoulterCat. No. 379503
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 29
Chapter 3 Target preparation on the Biomek FXP with Windows® XPEquipment, consumables, labware, and reagents required3
Biomek Span P1000 Pipette Tips (yellow box; pre-sterile, barrier, conductive)
Beckman CoulterCat. No. 987925
Biomek Span P50 Pipette Tips (pink box; pre-sterile, barrier)
Beckman CoulterCat. No. A21586
Bio-Rad Hard-Shell 96-well Plate(available in multiple colors)
Bio-RadCat. No. HSP-9631
96 Deep-Well Square Plate Thermo ScientificAB-0932
Table 6 Labware and materials used on the Biomek™ workstation deck (Continued)
Labware Supplier and Cat. No.
Labware image
30 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPEquipment, consumables, labware, and reagents required 3
OD Plate, UV E & K ScientificEK-25801
Lid, metal(arched, auto-sealing with P pads)
Bio-RadCat. No. MSL-2032 andP Pad Cat. No. MSP-1003
Hard-Shell Full-Height 96-Well Semi-Skirted PCR Plate • This consumable is required only if
using off-deck Applied Biosystems 2720 or Applied Biosystems 9700 Thermal cyclers
Bio-RadCat. No. HSS-9601
Table 6 Labware and materials used on the Biomek™ workstation deck (Continued)
Labware Supplier and Cat. No.
Labware image
Front view
Side view
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 31
Chapter 3 Target preparation on the Biomek FXP with Windows® XPEquipment, consumables, labware, and reagents required3
Plate, Costar Brand Serocluster Round Bottom Plate from Corning• Note: this consumable is required
only if using an off-deck Applied Biosystems 9700 or Applied Biosystems 2720 thermal cycle
VWR International Cat. No. 29442-392E&K Scientific Cat. No. EK 680568Corning Mfg Cat. No. 3795
The Applied Biosystems 9700 and the Applied Biosystems 2720 use the semi-skirted 96-well plates (Cat. No. HSS-9601). For use on the Biomek deck, the semi-skirted PCR plate must be stacked on a Costar brand Serocluster 96-well Round Bottom Microtitration plate as shown in the figure to the right.
Reagent Block Template(designed specifically for use with the Axiom 2.0 Reagent Kit)
Contact Thermo Fisher Scientific
Table 6 Labware and materials used on the Biomek™ workstation deck (Continued)
Labware Supplier and Cat. No.
Labware image
Template on reagent block. Metal posts on block circled in red.
32 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPEquipment, consumables, labware, and reagents required 3
Beckman Deep Well Titer Plate(polypropylene)
Beckman CoulterCat. No. 267007
Frame for reservoirs Beckman CoulterCat. No. 372795
Half reservoirHalf module, 75 mL capacity
Beckman CoulterCat. No. 372786
Table 6 Labware and materials used on the Biomek™ workstation deck (Continued)
Labware Supplier and Cat. No.
Labware image
Frame
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 33
Chapter 3 Target preparation on the Biomek FXP with Windows® XPEquipment, consumables, labware, and reagents required3
Reagent block, chilled to 4°C Beckman CoulterCat. No. A83054
Quarter Reservoirs• Quarter module, 40 mL capacity• Quarter module divided by width,
19 mL capacity each receptacle
Beckman Coulter
Cat. No. 372790 (40 mL)Cat. No. 372792 (19 mL)
24-Position Tube Rackwith one 11 mm tube insert in position A6.
Beckman CoulterCat. No. 373661 (rack)Cat. No. 373696 (insert)
Table 6 Labware and materials used on the Biomek™ workstation deck (Continued)
Labware Supplier and Cat. No.
Labware image
Metal posts on block circled in red.A1
Divided by width19 mL capacity
Undivided40 mL capacity
Tube insert A6
34 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPEquipment, consumables, labware, and reagents required 3
Adaptor, Deep Well Plate (installed on the Shaking Peltier)
This adaptor is typically installed by a Beckman Coulter field service technician during new system installation or a system upgrade. Ensure that you have one of these adaptors on the deck prior to running this assay.
Beckman CoulterCat. No. A83050
Zerostat Anti-static Gunand Ion-Indicator Cap
Milty Zerostat, Thermo Fisher Scientific Cat. No. 74-0014
Table 6 Labware and materials used on the Biomek™ workstation deck (Continued)
Labware Supplier and Cat. No.
Labware image
The metal block is the adaptor.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 35
Chapter 3 Target preparation on the Biomek FXP with Windows® XPEquipment, consumables, labware, and reagents required3
GeneTitan MC Instrument consumablesAll consumables for the GeneTitan MC Instrument are provided by Thermo Fisher Scientific. The following table provides guidance on the consumables that are shipped with the array plate.
IMPORTANT! All covers must have barcodes. Discard any cover without a barcode.
Table 7 Axiom™ GeneTitan™ MC Instrument consumables (Axiom 96-array plate)
Item Part number Image Information
Axiom 96-array plate, various designs
Varies, depending on array design.
Axiom 96-Array plate:• Comprised of 3 parts:
clear plastic cover, array plate, and blue array plate protective base.
• The clear plastic cover for the array plate protects the array plate during transport. Discard after opening pouch.
• The array plate must always be kept in the blue array plate protective base at all times.
• The blue array plate protective base in the package holds the array and protects it from damage or exposure to dust.
Note: Array plate is not included in the Axiom GeneTitan Consumables Kit.
Clear tray shipping cover (to be discarded)Array plate protective baseArray plate
1
2
3
1
2
3
36 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPEquipment, consumables, labware, and reagents required 3
Table 8 Axiom™ GeneTitan™ MC Instrument Consumables (from the Axiom™ Array GeneTitan™ Consumables Kit, Cat. No. 901606)
Item Part number Image Information
Scan Tray 900746 Box501006 Pouch
96-Plate Scan Tray:• Comprised of 3
parts: scan tray, black protective base, and a scan tray cover.
• The black scan tray protective base in the package protects the glass bottom of the scan tray from damage before it is loaded into the GeneTitan MC Instrument.
• The scan tray cover protects the contents in the scan tray and must be deionized before used. See Appendix E, "Deionization procedure for GeneTitan™ trays and covers" on page 307.
• Remove the black scan tray protective base before loading the scan tray with the scan tray cover into the GeneTitan MC Instrument.
• The Scan Tray must be loaded into the GeneTitan Instrument with the Scan Tray Cover only.
• Do not load the Scan Tray with the protective base still on.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 37
Chapter 3 Target preparation on the Biomek FXP with Windows® XPEquipment, consumables, labware, and reagents required3
Black Scan Tray Protective Base, shown without the Scan Tray with cover
• The black scan tray protective base in the package is used to protect the bottom of the scan tray glass from damage. The black scan tray is distinct from the blue array plate protective base and must not be used with the array plate.
• Remove and set aside the protective base from the scan tray before loading.
Scan Tray with cover, shown without the black protective base
• The GeneTitan scan tray must be loaded with the scan tray cover into the GeneTitan MC Instrument.
• Do not load the scan tray with the protective base.
Table 8 Axiom™ GeneTitan™ MC Instrument Consumables (from the Axiom™ Array GeneTitan™ Consumables Kit, Cat. No. 901606) (Continued)
Item Part number Image Information
38 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPEquipment, consumables, labware, and reagents required 3
GeneTitan 5 Stain Trays Kit
4249910 Kit501025 Tray
• The GeneTitan Stain Tray Kit comes with 5 stain trays packaged in zip-top bags to keep them free of dust.
• The GeneTitan stain trays are barcoded and the trays have separator walls that are flush with the frame of the stain tray, as shown by the yellow line and the yellow oval in the lower photo.
GeneTitan™ Stain and Scan Tray Cover
202757 • The GeneTitan stain and scan tray covers prevent evaporation of the stains in stain trays and the array holding buffer in the scan tray.
• All stain and scan trays must be placed in the GeneTitan MC Instrument with the GeneTitan stain tray cover.
• All tray covers must be deionized to remove static electricity prior to placing the cover on the tray.
• See the section "Deionization procedure for GeneTitan™ trays and covers" on page 307 for the anti-static procedure.
Table 8 Axiom™ GeneTitan™ MC Instrument Consumables (from the Axiom™ Array GeneTitan™ Consumables Kit, Cat. No. 901606) (Continued)
Item Part number Image Information
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 39
Chapter 3 Target preparation on the Biomek FXP with Windows® XPEquipment, consumables, labware, and reagents required3
Proper tray alignment and loading
Proper alignment and loading of plates, covers and trays is critical when using the GeneTitan MC Instrument. Each plate, cover and tray has 1 notched corner. The notched corner of plates, trays, covers and bases must be in vertical alignment with each other, and placed in position A1 per the Tray Alignment guide inside each GeneTitan MC Instrument drawer (Figure 9 and Figure 10 on page 42).
Note: Tip: Mark the notched corner of each plate, cover and tray with permanent marker to help ensure proper alignment and loading onto the GeneTitan MC Instrument.
Note: The instrument control software will display a warning if it detects a problem during the fluid dispense operations. The filters in the GeneTitan Wash A, Wash B and Rinse bottles should be replaced if the software displays such a warning. See Appendix F, ʺGeneTitan™ Multi-Channel Instrument Careʺ on page 313 for the
GeneTitan stain tray shown with the stain tray cover
Tray 501025Cover 202757
Hybridization Tray
900747 • After aliquoting the denatured hybridization ready samples into the hybridization tray, the tray should be immediately loaded into the GeneTitan MC Instrument with the barcode facing away from the operator, i.e., Barcode should be on the back side.
Table 8 Axiom™ GeneTitan™ MC Instrument Consumables (from the Axiom™ Array GeneTitan™ Consumables Kit, Cat. No. 901606) (Continued)
Item Part number Image Information
IMPORTANT! When running a multi-plate workflow, you must pay careful attention to the software prompts that tell you which side of the drawer to place or remove a plate/tray.
CAUTION! Take care not to damage the consumables or bend the blue cover posts or scan tray posts.
40 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPEquipment, consumables, labware, and reagents required 3
message displayed to the user and the procedure for replacing the filters.
Figure 9 Proper alignment and loading of plates, covers and trays in the GeneTitan MC Instrument
Plates and trays must be seated in this rectangular recess.
The notched corner of all plates, bases, and covers and must be seated in this corner of the drawer per
Tip: Mark the notched corner of each plate, cover and tray with permanent marker to help ensure proper alignment and loading.
Notched corner of array plate aligned with notched corner of blue base.
IMPORTANT! Remove the plastic protective shipping tray cover. 1
2
3
Clear tray shipping cover (to be discarded)Array plate protective baseArray plate
1
2
344
5
5
6
7
7
6
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 41
Chapter 3 Target preparation on the Biomek FXP with Windows® XPEquipment, consumables, labware, and reagents required3
Figure 10 Array plate with protective blue base and the hybridization tray aligned and properly loaded into drawer 6
Array plate with protective blue base
Hybridization tray
1
2
1 2
IMPORTANT! When you install the consumables, ensure that the fingers are retracted. Do not lay the consumables on top of the drawer fingers—this indicates that the instrument is not functioning correctly. Notify your field service engineer if the fingers do not retract automatically. You should place the trays into the instrument drawers when a drawer is fully extended by the instrument. The fingers are retracted when the drawer is open and are extended when the drawer is closed in order to restrain the consumable. See Figure 11 for an image showing the location of the tabs.
Figure 11 Photo identifying the location of drawer tabs
2
1
Drawer tab, or “finger” in back.
Drawer tab, or “finger” on right side.
1
2
42 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPEquipment, consumables, labware, and reagents required 3
Stain trays and covers
Labeling GeneTitan hybridization and reagent traysWhen preparing the hybridization and reagent trays to be loaded onto the GeneTitan MC Instrument, you will need to mark each tray in a way that identifies its contents.
Proper labeling for hybridization trays and reagent trays is described in:
• ʺLabeling for hybridization traysʺ, below
• ʺLabeling for stain traysʺ on page 44
Labeling for hybridization trays
You may label the hybridization tray on the front part of the short side of the tray, next to the notch at the left, as shown in Figure 13. The proper section for labeling is closest to the notched corner, corresponding to the A1 and B1 wells.
IMPORTANT! Always place the flat side of the cover against the stain tray.
Figure 12 Placement of covers on trays
Correct placement of cover on stain tray. Incorrect placement of cover on stain tray.
IMPORTANT! It is critical that you write only on the proper locations of the proper sides of hybridization and stain trays. Do NOT write in any other location, as this can interfere with sensors inside the GeneTitan MC Instrument and result in experiment failure. To ensure proper placement of lids onto stain trays, and trays onto the GeneTitan MC Instrument, you can also mark the notched corner of the trays and lids.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 43
Chapter 3 Target preparation on the Biomek FXP with Windows® XPEquipment, consumables, labware, and reagents required3
Labeling for stain trays
You may label the stain trays on the left side of the front of the tray as shown in Figure 14. The correct side is closest to the notched corner, corresponding to the A1 through C1 wells.
Figure 13 Labeling GeneTitan hybridization trays
CAUTION! Writing on the wrong side of the hybridization tray may interfere with the operation of sensors in the GeneTitan MC Instrument.
IMPORTANT! Do not confuse hybridization trays with stain trays.
Do NOT label trays on the long side of the tray.
Notched corner of the hybridization tray should face the front.
Label the hybridization tray in this area.
1
2
3
1 2 3
44 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPEquipment, consumables, labware, and reagents required 3
Figure 14 Labeling GeneTitan stain tray (stain tray shown with lid)
Do NOT label trays on the long side of the tray.
Notched corner of the stain tray should face the front.
Label the stain tray here.
1 2 3
1
2
3
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 45
Chapter 3 Target preparation on the Biomek FXP with Windows® XPEquipment, consumables, labware, and reagents required3
Loading tray consumables onto the GeneTitan™ MC Instrument
Loading, or installing, the trays and plates onto the GeneTitan MC Instrument is a simple procedure, but there are certain precautions that you must take in order to ensure an error-free procedure. The following figures show you how to do it. See Figure 15 through Figure 18.
Figure 15 Scan tray with cover loaded in drawer 2
IMPORTANT! When you load the plates, or trays, insert them under the tabs, or fingers, that may protrude into the stage. Confirm that the tray is not resting on these fingers. See Figure 108 on page 211.
Do NOT load the protective black base packaged with the scan tray.
46 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPEquipment, consumables, labware, and reagents required 3
Figure 16 Stain 1 tray and Ligation tray loaded in drawer 3
Figure 17 Stain 2 tray and Stabilization tray loaded in drawer 4
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 47
Chapter 3 Target preparation on the Biomek FXP with Windows® XPEquipment, consumables, labware, and reagents required3
Reagent block template
The Axiom™ 2.0 Reagent Kit template fits precisely onto the top of the chilled reagent block, and is held in place by the metal posts on the block (Figure 19). Using this template will help ensure the proper placement of reagent tubes onto the block for each method.
Reservoir labels The reservoir labels are stick-on labels for the modular reagent reservoir frames for the different stages of the Automated workflow. These stick-on labels are color-coded to match the colors found on the caps of the reagent tubes in the Axiom 2.0 Reagent Kit. Using these labels helps ensure the proper placement of reservoirs and reagents for each method.
There are 4 reservoir holders used in the Axiom 2.0 method. Three of these will have templates on 2 sides and the remaining reservoir holder will have a template on 1 side for a total of 7 templates.
Figure 18 Stain 1 tray loaded in drawer 5
Figure 19 Axiom 2.0 Reagent Template for the reagent block
48 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPEquipment, consumables, labware, and reagents required 3
Remove the protective surface from the back of the label and place on the reservoir frames as directed in the Figure 20 through Figure 23.
Reservoir frame 1
Reservoir frame 2
Figure 20 Reservoir frame 1 for Windows® XP users
Figure 21 Reservoir frame 2 for Windows® XP users
Side A
Side B
Side A
Side B
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 49
Chapter 3 Target preparation on the Biomek FXP with Windows® XPEquipment, consumables, labware, and reagents required3
Reservoir frame 3
Reservoir frame 4
Related Biomek FXP Target Prep Express documentation
The following user manuals are installed at the same time as the Biomek FXP Target Prep Express software (Start All Programs Beckman Coulter Manuals). See these for troubleshooting the Biomek workstation.
• Biomek® Liquid Handler User’s Manual, Beckman Coulter Pub. No. 987834
• Biomek® Software User’s Manual, Beckman Coulter Pub. No. 987835
Figure 22 Reservoir frame 3 for Windows® XP users
Figure 23 Reservoir frame 4 for Windows® XP users
Side A
Side B
Side A
50 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 1: DNA Amplification 3
Stage 1: DNA Amplification
Note: For this protocol, the term samples includes the positive control.
Duration Note: A 22–24 hours incubation is required at the end of this stage.
Equipment, consumables, labware, and reagents required
Equipment and labware required
IMPORTANT! Before proceeding to DNA Amplification, do the gDNA preparation described in Chapter 2, ʺGenomic DNA preparation and requirementsʺ on page 13.
Table 9 Time required for "Stage 1: DNA Amplification"
Activity Time
Hands-on time ~30 minutes
Biomek FXP Target Prep Express ~19 minutes
Incubation 23 hours
Total time ~24 hours
Table 10 Equipment and labware required
Quantity Item
As required Adhesive seals for plates
1 Benchtop cooler, chilled to –20°C
As required Kimwipes laboratory tissues
1 Marker, fine point, permanent
1 Mini microcentrifuge (microfuge with microtube rotor)
1 Oven (must maintain a constant temperature of 37°C for at least 23 hours with a temperature accuracy of 1°C)• >3 array plates per week—we recommend using the BINDER ED 56• 3 array plates per week—OK to use the GeneChip Hybridization Oven
or the BINDER ED 56
1 Plate centrifuge
1 Vortex
Biomek workstation labware
1 box of each Barrier pipette tips:• Biomek Span P1000 (yellow)• Biomek AP96, P250 (aqua)
2 Plate, Bio-Rad Hard-Shell PCR 96-well
2 Plate, deep well titer
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 51
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 1: DNA Amplification3
Reagents required
1 Reagent block, chilled to 4°C
3 Reservoir, quarter module (40 mL)
2 Reservoir, half module (75 mL)
Table 11 Reagents required for DNA Amplification
Axiom 2.0 Reagent Kit Module
Axiom 2.0 Denat Soln 10X
Module 1, –20°C
Axiom 2.0 Neutral Soln
Axiom 2.0 Amp Soln
Axiom 2.0 Amp Enzyme
Axiom Water
IMPORTANT! The Axiom 2.0 Assay is compatible with only reagents from an Axiom Reagent Kit. These reagents are not interchangeable with reagents from other Applied Biosystems reagent kits, such as SNP 6.0, DMET Plus, etc. The new Axiom 2.0 Reagent Kit contains a different Module 1 than the original Axiom Reagent Kit; however, Modules 2, 3, and 4 are identical between both versions and may be used from either kit for this assay. Only use new Module 1 from the Axiom 2.0 Reagent Kit for the Axiom 2.0 Assay.
Table 10 Equipment and labware required (Continued)
Quantity Item
52 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 1: DNA Amplification 3
1. Perform the pre-run checklist
Check the water and waste containers
1. If necessary, fill the system water container using deionized water (no need for ultra-pure water).
2. If necessary, empty the system waste bottle.
Turn on the Biomek FXP Target Prep Express
1. Power on the workstation.
2. Ensure that all of the peripherals are powered on.
• Watlow temperature controllers
Control the Static Peltier (Pelt_1) and the Shaking Peltier (SPelt_96); no additional power supply.
• Thermal cycler:
Bio-Rad PTC-200 or Biometra TRobot 96 if present on the Biomek workstation deck. Otherwise, a stand-alone thermal cycler can be used.
3. Launch the Biomek Software by double-clicking the Biomek Software icon on the desktop .
You can also open Start All Programs Beckman Coulter Biomek Software.
Close the thermal cycler (on deck thermal cycler)If your Biomek workstation includes a PTC200 or a Biometra TRobot 96, the lid may remain open upon startup. You must close the thermal cycler lid prior to homing the axes or starting a method.
The lid of the PTC-200 opens automatically when the device is powered up. While the lid of the TRobot does not power up automatically, it is possible that the lid may be open from a previous run or at the beginning of the next run.
Note: See page 12 of the setup guide and user’s manual for Biometra TRobot on the Biomek FXP. After following the instructions to close the thermal cycler lid, proceed to ʺHome All Axesʺ on page 56.
CAUTION! Close the thermal cycler lid:
• If it remains open after you have powered on the workstation.
• If the lid is up before you home the axes or before you begin a method.
If not closed, the MC Pod may collide with the thermal cycler lid and damage the instrument.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 53
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 1: DNA Amplification3
Closing the thermal cycler lid on the PTC-200
1. Open Instrument Device Editor.
2. Open the Device drop-down menu and select PTC200Left.
3. Click Action Commands.
4. Select the following (Figure 24 on page 55):
a. In the Actions box, select Close.
b. In the Open/Close box, select Without plate.
5. Click Close Lid.
A Status window is displayed while the lid is being closed (Figure 25).
6. Click OK when the Command executed prompt is displayed (Figure 25).
7. Click Cancel; then click Close.
IMPORTANT! It is critical that you select Without plate in the Open/Close box. If a plate is present, remove it now.
54 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 1: DNA Amplification 3
Figure 24 Closing the thermal cycler lid
Figure 25 Closing PTC lid prompts.
1
3
2
1
2
3
Select Close in the Actions box.
Select Without plate in the Open/Close box. This is very IMPORTANT to avoid damage to the thermal cycler!
Click Close Lid.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 55
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 1: DNA Amplification3
Home All AxesThis procedure will home all axes and prime the fluidics lines.
1. Open Instrument Home All Axes.
2. Ensure all conditions in the Warning prompt Figure 26-A are met, then click OK.
An icon instructing you to stop and wait while the instrument homes is displayed (Figure 26-B).
3. When:
a. the Warning prompt in Figure 27-A is displayed, confirm that no tips are loaded in the Span-8 Pod, and click OK.
The lines for the Span-8 tips are primed and the next prompt shown in Figure 27-B is displayed.
b. the intake (syringes and tubing) for the Span-8 tips is clear of bubbles, click OK.
Figure 26 Homing All Axes
Figure 27 Prompts displayed for priming the Span-8 pod fluidic lines
A B
A B
56 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 1: DNA Amplification 3
2. Thaw and prepare the reagents and Sample Plate
Thaw and prepare the reagents and Sample Plate
To thaw and prepare the reagents:
1. Thaw the Sample Plate (containing 200 ng of gDNA at 10 ng/µL or 100 ng of gDNA at 5 ng/µL depending on the Axiom array plate to be used) on the benchtop at room temperature and centrifuge.
Note: Do not place a frozen Sample Plate directly on the workstation deck.
2. Thaw the following reagents on the benchtop at room temperature.
• Axiom 2.0 Denat Soln 10X
• Axiom 2.0 Neutral Soln
• Axiom 2.0 Amp Soln
• Axiom Water
Leave the Axiom 2.0 Amp Enzyme in the freezer until ready to use.
Note: Allow ~1 hour for Axiom 2.0 Amp Soln to thaw on the benchtop at room temperature. If the solution is not completely thawed after 1 hour, vortex briefly and return to the benchtop to complete thawing. The bottles can also be thawed in a dish with Millipore water. The Axiom 2.0 Amp Soln must be thoroughly mixed before use.
3. Vortex all reagents (except Axiom 2.0 Amp Enzyme), then place at room temperature.
• Vortex the Axiom 2.0 Amp Soln and Axiom 2.0 Neutral Soln for 30 seconds to thoroughly mix.
• Vortex and centrifuge the Axiom 2.0 Denat Soln 10X before placing on the deck.
• For the Axiom 2.0 Amp Enzyme, just before placing on the deck gently flick the tube 3 times to mix and centrifuge.
4. Preheat the Oven to 37°C.
We recommend using one of these ovens:
• BINDER ED 56
• GeneChip™ Hybridization Oven (turn rotation on to 15 rpm)
3. Run the DNA Amplification step
To run the DNA Amplification step:
1. Open Project Open Project Axiom 2.0 Target Prep and click OK.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 57
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 1: DNA Amplification3
2. Open File Open to display the Open Method window.
Or click the Open Method icon .
3. Select Axiom 2.0 Target Prep and click OK.
4. At the top of the main window (Figure 28-A), click the Run button to open the Axiom™ Target Prep window.
5. In the Axiom Target Prep window (Figure 28-B):
a. Select DNA Amplification.
b. Click OK.
The Deck Layout for DNA Amplification is displayed (Figure 29 on page 59).
The Axiom 2.0 Target Prep project folder must be displayed in this menu.
1
1
Figure 28 Opening the Axiom Target Preparation methods window
A B
58 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 1: DNA Amplification 3
6. Place the labware and reagents on the deck as directed in the following figures and table:
• Figure 29 on page 59—deck layout
• Figure 30 on page 60—location names of empty deck positions
• Table 12 on page 60—table detailing the labware, reagent and modular reservoir placement for the deck layout
• Figure 31 on page 61—reagent block
IMPORTANT! Axiom 2.0 Amp Enzyme—Immediately prior to placing on the reagent block, gently flick the tube with your finger 2 to 3 times to mix; then centrifuge. Do not vortex.
Figure 29 Deck layout window for the DNA Amplification method
IMPORTANT: No tips should be loaded onto the movable arms referred to as the Span-8 (right) and the Multi-Channel (MC; left) pods.
MC pod uses the pipette tips in this position.
Span-8 pod uses the pipette tips in this position.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 59
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 1: DNA Amplification3
Figure 30 Empty deck positions
Table 12 Labware and reagent locations on the deck for the DNA Amplification method
Position on deck
Labware Reagent or samples
TL1 Biomek AP96 – P250 Pipette Tips (aqua) —
P1 Beckman Deep Well Titer Plate gDNA samples
P4 Bio-Rad Hard-Shell 96-well plate (any color) —
P10 Reservoirs in frame:• Quarter module (1)• Quarter module (2)• Quarter module (3)
Pour Axiom 2.0 Neutral Solution into Reservoir 2
P11 Reservoirs in frame:• Half module (1)• Half module (2)
• Pour Axiom Water into reservoir 1• Pour Axiom 2.0 Amp Soln into reservoir 2
Pelt_1 Reagent block, chilled to 4°C See Figure 31.
P14 Biomek Span P1000 Pipette Tips (yellow) —
P15 Bio-Rad Hard-Shell 96-well plate (any color)
SPelt96_1 Beckman Deep Well Titer Plate —
1Empty
Reservoir
2Axiom
2.0 Neutral
Soln
3Empty
Reservoir
1
Axiom Water
2
Axiom 2.0 Amp Soln
60 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 1: DNA Amplification 3
7. Check the deck layout to ensure that all labware and reagents are in the proper locations.
Note: If the physical deck does not match the Deck Layout and Confirmation window exactly (Figure 29 on page 59), either modify the physical deck to match exactly or choose Abort in the Deck Layout and Confirmation window.
8. Click OK.
The system flushes the Span-8 fluidics system. Observe the lines and syringes for air bubbles.
Figure 31 Placement of reagents on chilled reagent block for the DNA Amplification step
Important:
• Position all plates and the chilled reagent block with A1 in the upper left corner of the frame.• Centrifuge all reagent tubes prior to placing in block to avoid loss of solution volume to the cap and sides of the tube.• Press reagent tubes into the block to ensure that they are fully seated.
Axiom 2.0Amp
Enzyme
Axiom 2.0 Denat Soln
10X
Reagent block with template.
Only the positions used for this method are shown.
Flick and centrifuge the Amp Enzyme before placing in block.
Diagram of reagent block without template
A1
A1
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 61
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 1: DNA Amplification3
9. At the prompt to repeat the Span-8 fluidics system flush (Figure 32):
• Click No if no air bubbles are present.
• Click Yes if air bubbles are present. Repeat the flush until no air bubbles are present.
The DNA Amplification step runs until the Amplification Master Mix has been added to the Sample Plate. Once complete, the instructions and prompt shown in Figure 33 are displayed.
• Follow the instructions for the Sample Plate (see also ʺUser interventionʺ below).
• Follow the instructions for clearing the workstation deck.
User intervention
1. Remove the Sample Plate.
2. Blot the top of the plate with a Kimwipes laboratory tissue to remove any droplets that may be present.
3. Tightly seal the plate.
4. Vortex and centrifuge the plate.
5. Place in a preheated oven and incubate at 37°C for 23 1 hour.
Note: If using a GeneChip™ Hybridization Oven, place the plate on the bottom of the oven. Plates do not rotate.
6. After 22–24 hours of incubation, do one of the following:
• Proceed directly to ʺStage 2: Fragmentation and Purificationʺ on page 65.
• Tightly seal and store the amplified samples at –20°C.
Figure 32 Flushing the Span-8 fluidics system to purge air bubbles
Figure 33 Instructions for clearing the workstation deck
IMPORTANT:
• Seal the Sample Plate before placing in the oven.• Always discard the used multichannel pipette tips in position P3.• Always store the reagent block at 4°C.
62 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 1: DNA Amplification 3
Summary of DNA Amplification
Stage 1: Amplification
Stage 1: Amplification Page 1
Transfer Denat Soln 1X to Sample Plate
Prepare Denat Soln 1X & Distribution Plate
Span-8
Incubate on Deck10 mins @ RT
Denat Soln 10X Axiom Water
[96] 488 μL 96] 4392 μL
(4°C) (RT)D1 Reservoir
D1 Plate
30 μL/well
D1 Plate Sample Plate
20 μL/well
MC
Continued on AmplificationPage 2
Fill Neutral Soln Plate
N1 Reservoir N1 Plate
140 μL/well
[96] FORMAT TRANSFER MIX MOVE
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 63
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 1: DNA Amplification3
Span-8MC
Continued from AmplificationPage 1
Prepare Amplification Master Mix & Distribution Plate
Amp Soln Amp Enzyme
[96] 26374 μL [96] 586 μL
(RT) (4°C)MM Reservoir
Amp Mastermix Plate
255 μL/well
Stage 1: Amplification Page 2
Stage 1: Amplification
Transfer Neutral Soln to Sample Plate
N1 Plate Sample Plate
130 μL/well
Incubate Sample PlateOffline for 23 ±1hrs @ 37°C
Transfer Amplification Master Mix to Sample Plate
Amp Mastermix Plate Sample Plate
230 μL/well
[96] FORMAT TRANSFER MIX MOVE
64 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 2: Fragmentation and Purification 3
Stage 2: Fragmentation and Purification
Note: Purification is done by precipitation (See Step ʺ4. Precipitationʺ on page 75).
Duration
Equipment, consumables, labware, and reagents required
Equipment and labware required
Table 13 Time required for "Stage 2: Fragmentation and Purification"
Activity Time
Hands-on time ~25 minutes~50 minutes if frozen DNA from Step 1
Biomek FXP Target Prep Express• Deactivation incubation—20 minutes to deactivate the
amplification reaction and 20 minutes to equilibrate to the fragmentation temperature
• Fragmentation incubation—30 minutes
~1:35 hours
Total time 2:00 to 2:25 hours
Table 14 Equipment, consumables, and labware required
Quantity Item
As required Adhesive seals for plates
1 Benchtop cooler, chilled to –20°C
1 Freezer, –20°C
1 Ice bucket, filled with ice
As required Kimwipes laboratory tissues
1 Marker, fine point, permanent
1 Mini microcentrifuge (microfuge with microtube rotor)
1 Oven, preheated to 37°C
1 Plate centrifuge
1 Vortex (for plates and microtubes)
Biomek workstation labware
1 box of each Barrier pipette tips:• Biomek AP96, P250 (aqua)• Biomek Span P250 (green)• Biomek Span P1000 (yellow)
3 Plate, Bio-Rad Hard-Shell PCR 96-well
1 96 Deep-Well Square Plate
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 65
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 2: Fragmentation and Purification3
Reagents required
1. Perform the pre-run checklist
The following actions are the same as described under ʺ1. Perform the pre-run checklistʺ on page 53. Refer back to this step for details. Some or all of these steps may not be required depending upon the current state of the Biomek workstation.
To perform the pre-run checklist:
1. Power on the Biomek FXP Target Prep Express and all peripherals.
2. Check the water and waste containers; replenish or empty as required.
3. Launch the Biomek Software.
4. If applicable, close the thermal cycler lid (on page 53).
5. Home all axes (on page 56).
1 Reagent block, chilled to 4°C
3 Reservoir, quarter module
1 Reservoir, half module
Table 15 Reagents required for the Fragmentation method
Reagent Module
From the Axiom 2.0 Reagent Kit
Axiom Frag Enzyme (leave at –20°C until ready to use)Module 2-1, –20°C
Axiom 10X Frag Buffer
Axiom Precip Soln 2
Axiom Frag DiluentModule 2-2, 2–8°C
Axiom Frag Rxn Stop
Axiom Precip Soln 1
User-supplied
Isopropanol, 99.5% 96 samples: 65 mL
Table 14 Equipment, consumables, and labware required (Continued)
Quantity Item
66 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 2: Fragmentation and Purification 3
2. Thaw and prepare the amplified DNA samples and reagents
Thaw and prepare the amplified DNA Sample Plate
If the plate of amplified samples is frozen:
1. Place the deep well plate in a small water bath.
For example, pour Millipore water into a small tray. Place the frozen plate on the water in the tray.
2. Leave the plate in the water bath for ~50 minutes until all wells have thawed.
3. Centrifuge at 1,000 rpm for 30 seconds.
4. To avoid cross-contamination of wells during vortexing:
a. Remove the seal and blot the top of the plate with a Kimwipes laboratory tissue.
b. Tightly reseal the plate with a fresh seal.
5. Vortex the plate for 30 seconds to thoroughly mix.
6. Centrifuge at 1,000 rpm for 30 seconds.
Thaw and prepare the reagents
1. Thaw the following reagents on the benchtop at room temperature.
• Axiom 10X Frag Buffer
• Axiom Precip Soln 2
2. Vortex all reagents (except Axiom Frag Enzyme), then place on ice.
• Vortex and centrifuge Axiom Precip Soln 2 before placing onto deck.
• For the Axiom Frag Enzyme: Leave at –20°C until ready to use. Just before placing on the deck gently flick the tube 3 times to mix and centrifuge.
3. Run the Fragmentation step
To open the Target Preparation methods window:
1. Open Project Open Project Axiom 2.0 Target Prep and click OK.
2. Click the Open Method icon, select Axiom 2.0 Target Prep, and click OK.
3. Click the Run button.
4. Select Fragmentation, then click OK.
The deck layout for Fragmentation is displayed (Figure 34).
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 67
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 2: Fragmentation and Purification3
5. Place the labware and reagents on the deck as directed in the following figures and table:
• Figure 34 on page 68—deck layout
• Figure 35 on page 69—location names of empty deck positions
• Table 16 on page 69—table detailing the labware, reagent and modular reservoir placement for the deck layout
• Figure 36 on page 70—reagent cold block
IMPORTANT! Remove the seal from the Sample Plate before placing on the deck.
Figure 34 Deck layout for the Fragmentation method
68 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 2: Fragmentation and Purification 3
Figure 35 Empty deck positions
Table 16 Labware and reagent locations on the deck for the Fragmentation step
Position on deck
Labware Reagent or samples
TL1 Biomek AP96 – P250 Pipette Tips (aqua) —
P4 Bio-Rad Hard-Shell 96-well plate —
P5 Bio-Rad Hard-Shell 96-well plate —
P6 Bio-Rad Hard-Shell 96-well plate —
P7 Beckman Deep Well Titer Plate Amplified gDNA
P8 96 Deep-Well Square Plate —
P10 Reservoirs in frame:• Quarter module (1)• Quarter module (2)
• Pour Axiom Frag Rxn Stop into reservoir 1• Pour Axiom 10X Frag Buffer into reservoir 2
P11 Reservoirs in frame:• Half module (1)• Quarter module (2)
• Pour Isopropanol into reservoir 1• Pour Axiom Precip Soln 1 into reservoir 2
Pelt_1 Reagent block, chilled to 4°C See Figure 36 on page 70.
P13 Biomek Span P250 Pipette Tips (green)
P14 Biomek Span P1000 Pipette Tips (yellow) —
1AxiomFragRxnStop
2Axiom10X Frag
Buffer
1
Isopropanol
2AxiomPrecipSol 1
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 69
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 2: Fragmentation and Purification3
6. Check the deck layout to ensure that the labware, reagents and samples are in the proper locations.
Note: If the physical deck does not match the Deck Layout and Confirmation window exactly (Figure 34 on page 68), either modify the physical deck to match exactly or choose Abort in the Deck Layout and Confirmation window.
7. Click OK to continue.
The system will automatically flush the Span-8 fluidics system. Observe the lines and syringes for air bubbles.
8. At the prompt to repeat the Span-8 fluidics system flush:
• Click No if no air bubbles are present.
• Click Yes if air bubbles are present. Repeat the flush until no air bubbles are present.
Figure 36 Placement of reagents on chilled reagent block for the Fragmentation step
IMPORTANT:
• Always position the chilled reagent block with A1 in the upper left corner of the frame.• Centrifuge all reagent tubes prior to placing in block to avoid loss of solution volume to the cap and sides of the tube.• Press reagent tubes into the block to ensure that they are fully seated.
Axiom Precip Soln 2
Axiom Frag
Diluent
Axiom Frag
Enzyme
Reagent block with template.
Only the positions used for this method are shown.
Diagram of reagent block without template
Axiom Frag Enzyme:
Flick to mix 3 tomes and centrifuge immediately prior to placing on the chilled reagent block.
A1
A1
70 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 2: Fragmentation and Purification 3
The Fragmentation step begins. The Sample Plate is incubated at 65°C to inactivate amplification.
9. If you selected Prompt for manual DNA quantitation in the default software settings, you will then be prompted to remove an aliquot of each sample for a optional quantitation process control. The plate will remain at 40°C until the aliquots have been collected. Remove 4 µL aliquots from each well into a 96 well PCR plate and set aside for later quantitation (e.g., using the Quant-iT™ PicoGreen® dsDNA Kit from Life Technologies). Immediately continue sample processing by placing the amplification reactions back onto the shaking peltier and clicking OK at the prompt shown in Figure 37.
If Prompt for manual DNA quantitation was not selected, the box in Figure 37 will not appear.
Note: Remain near the Biomek FXP Target Prep Express if you are going to remove aliquots for quantitation. Avoid leaving the samples at 40°C for a long period of time.
10. Once the Fragmentation Complete message box appears (Figure 38). Follow the instructions in the message box discarding used labware and tips, storing unused tips and storing the cold block at 4°C. Click OK when ready to continue and proceed to Step ʺ4. Precipitationʺ on page 75.
Figure 37 Prompt for manual DNA quantitation
Figure 38 Fragmentation complete message
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 71
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 2: Fragmentation and Purification3
Summary of Fragmentation
Stage 2: Fragmentation
Span-8MC
Stage 2: Fragmentation Page 1
Inactivate DNA Amplification Rxn
Sample Plate
Incubate:
1) 20 mins @ 65°C
2) 20 mins @ 40°C
SPelt
Fill Fragmentation Buffer Plate
FragB Reservoir FragB Plate
56 μL/well
Transfer Fragmentation Buffer to Sample Plate
Sample Plate
45.7 μL/well
FragB Plate
Prepare Fragmentation Reagent & Distribution Plate
Frag Di luent Frag Enzyme
[96] 1551.4 μL [96] 150.6 μL
(4 oC)
(4°C)Frag Enzyme Tube
FragR Plate
16.5 μL/well
((((((((((((((((((((((((((((4 444444 44444 444 ooooooCCCCCCCCCCCCCC))))))))))))))))))oCCC))))))
Continued on FragmentationPage 2
(4°C)
[96] FORMAT TRANSFER MIX MOVE
72 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 2: Fragmentation and Purification 3
Stage 2: Fragmentation Page 2
Stage 2: Fragmentation
Span-8MC
Continued from FragmentationPage 1
Incubate on Deck
30 mins @ 40oC
Transfer Fragmentation Reagent to Sample Plate
Sample Plate
11.3 μL/well
FragR Plate
Fill Stop Solution Plate
Stop Reservoir Stop Plate
29 μL/well
Prepare Prepitation Soln & Distribution Plate
Precip Soln 2 Precip Soln 1
[96] 192 μL
(4°C)
Precip Plate
240 μL/well
Continued on FragmentationPage 3
[96] FORMAT TRANSFER MIX MOVE
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 73
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 2: Fragmentation and Purification3
Stage 2: Fragmentation
Span-8MC
Stage 2: Fragmentation Page 3
Continued from FragmentationPage 2
Transfer Stop Solution to Sample Plate
Stop Plate Sample Plate
19 μL/well
Transfer Precipitation Solution to Sample Plate
Precip Plate Sample Plate
Plate Contents
Transfer Isopropanol to Precipitation Plate
Iso Reservoir Precip Plate
600 μL/well
Offline PrecipitationSee user guide
Transfer Precipitation Solution to Sample Plate
Sample Plate Precip Plate
Plate Contents
[96] FORMAT TRANSFER MIX MOVE
74 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 2: Fragmentation and Purification 3
4. Precipitation To freeze the samples:
1. Remove the Precipitation Plate from the deck.
2. Blot the top of the plate with a Kimwipes laboratory tissue and seal tightly.
3. Place the plate in a –20°C freezer overnight to precipitate.
4. Return to the Biomek workstation and clear the deck.
5. Centrifuge and dry pellets
Note: Keep the centrifuge ready at 4°C.
To centrifuge and dry the pellets:
1. Turn the oven on and preheat to 37°C.
Use an oven that can sustain a constant temperature of 37°C and has a temperature accuracy of 1°C (we recommend the BINDER ED 56). If processing 3 or fewer array plates, you can use a GeneChip Hybridization Oven.
2. Centrifuge the plate for 40 minutes at 4°C at 3,200 x g (4,000 rpm for the Eppendorf 5810R centrifuge with the rotor configuration described in the Axiom™ 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP Site Preparation Guide).
3. Immediately after the 40 minutes centrifugation period, empty the liquid from the plate as follows:
a. Remove the seal.
b. Invert the plate over a waste container and allow the liquid to drain.
c. While still inverted, gently press the plate on a pile of Kimwipes laboratory tissues on a bench and leave it for 5 minutes.
4. Turn the plate right side up and place in an oven for 20 minutes at 37°C to dry.
Note: If using a GeneChip Hybridization Oven, place the plate on the bottom of the oven. Plates do not rotate.
5. Do one of the following:
• Proceed directly to ʺStage 3: Resuspension and hybridization preparationʺ on page 76, even if some droplets of liquid remain. Leave the Sample Plate at room temperature. It is helpful to begin preparing reagents for Stage 3 while centrifuging and drying pellets.
• Tightly seal the plate and store at –20°C.
CAUTION! During this step, handle the plate gently to avoid disturbing the pellets. Do not bump or bang the plate.
WARNING! Use rotor buckets with a soft rubber bottom to ensure that the deep well plates do not crack. Do not use buckets where the plates sit directly on a metal or hard plastic bottom, such as the A-4-62 rotor with a WO-15 plate carrier (hard bottom) for the Eppendorf 5810R centrifuge. Use of hard bottom plate carriers may result in cracked plates, loss of sample, unbalanced centrifugation, damage to the instrument and possible physical injury.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 75
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 3: Resuspension and hybridization preparation3
Stage 3: Resuspension and hybridization preparation
Duration Resuspension and hybridization preparation
Equipment, consumables, labware, and reagents required
Equipment and labware required
Table 17 Time required for Resuspension
Activity Time
Hands-on time 15 minutes
Frozen pellet equilibration to room temperature 1.5 hours
Biomek FXP Target Prep Express 40 minutes
Table 18 Equipment, consumables, and labware required
Quantity Item
As required Adhesive seals for plates
1 Benchtop cooler, chilled to –20°C
1 Ice bucket, filled with ice
1 Marker, fine point, permanent
1 Mini microcentrifuge (microfuge with microtube rotor)
1 Plate centrifuge, at room temperature
1 Vortex
Biomek workstation labware
1 box of each Barrier pipette tips:• Biomek Span P50 (pink)• Biomek AP96, P250 (aqua)• Biomek Span P250 (green)• Biomek Span P1000 (yellow)
5 platesor:
Bio-Rad Hard-Shell PCR 96-well (HSP-9631)For on-deck thermal cycling or when using the PTC-0240/PTC-200 off-deck thermal cycler
6 plates for off deck
cycling with Applied
Biosystems thermal cyclers
4 Bio-Rad (HSP-9631) Hard-Shell PCR 96-well Plate and 1 HSS-9601 Hard-Shell Full-Height 96-Well Semi- Skirted PCR Plate1 Plate, Costar Brand Serocluster round bottom plate
For off-deck thermal cycling using the Applied Biosystems 9700 or Applied Biosystems 2720 thermal cycler.Note: The HSS-9601 plate stacked on the Costar brand serocluster round-bottom plate should only be used on position P2 on the Biomek FXP deck. See Figure 46 on page 92.
1 Plate, OD
76 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 3: Resuspension and hybridization preparation 3
Reagents required
1. Preparing frozen pellets and Axiom Resusp Buffer
The equilibration of resuspension buffer and pellets to room temperature (18°C to 25°C) is very critical for the success of the Axiom 2.0 assay. When either is cooler than room temperature, pellets may not resuspend completely. This may result in compromised assay performance. Note following guidelines on how to work with plates with fresh, cold or frozen pellets:
Pellets:
• Plates with fresh pellets can be kept at room temperature for up to 1 hour if proceeding with the resuspension and hybridization protocol within an hour.
• Plates with fresh pellets that are not processed within an hour can be transferred to a refrigerator (2-8°C) for few hours only if processed within a day. However, it is critical to equilibrate the plate to room temperature for at least 30 minutes before proceeding with the resuspension and hybridization protocol.
• Plates with frozen pellets (e.g., on Day-5 of 8-plate workflow) must be pre-equilibrated at room temperature for at least 1.5 hour before proceeding with the resuspension and hybridization protocol.
1 Reagent block, chilled to 4°C
1 Reservoir, 19 mL (quarter module, divided)
3 Reservoir, 40 mL (quarter module)
Table 19 Reagents required for Resuspension and Hybridization
Reagent Module
From the Axiom 2.0 Reagent Kit
Axiom Hyb Buffer Module 2-1, –20°C
Axiom Hyb Soln 1
Axiom Resusp Buffer Module 2-2, 2–8°C
Axiom Hyb Soln 2
Other reagents required
Nuclease-free Water, ultrapure MB grade (Thermo Fisher Scientific, Cat. No. 71786; for OD and gel plate preparation)
To fill line of divided reservoir
TrackIt Gel Loading Buffer, diluted 1,000-fold(see Appendix B, "Fragmentation quality control gel protocol" on page 292 for dilution instructions.)
13 mL
Table 18 Equipment, consumables, and labware required (Continued)
Quantity Item
IMPORTANT! The pellets and the resuspension buffer must be at room temperature before proceeding with this step.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 77
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 3: Resuspension and hybridization preparation3
Resuspension Buffer:
• Resuspension buffer when pulled out from Module 2-2 at 2-8°C, needs at least 1 hour to equilibrate to room temperature.
2. Perform the pre-run checklist
The following actions are the same as described under ʺ1. Perform the pre-run checklistʺ on page 53. Refer back to this step for details. Some or all of these steps may not be required depending upon the current state of the Biomek workstation.
1. Power on the Biomek FXP Target Prep Express and all peripherals.
2. Check the water and waste containers; replenish or empty as required.
3. Launch the Biomek Software.
4. If applicable, close the thermal cycler lid (on page 53).
5. Home all axes (on page 56).
3. Thaw and prepare the reagents
Thaw and prepare the reagents
1. Thaw Axiom Hyb Soln 1 and warm Axiom Resusp Buffer on the benchtop at room temperature.
2. Vortex the Axiom Resusp Buffer and the Axiom Hyb Buffer.
3. Vortex and centrifuge Axiom Hyb Soln 1 and Axiom Hyb Soln 2.
4. Run the Resuspension and Hybridization Preparation step
Note: We strongly recommend that you run 2 quality process controls during this step:
• A gel to verify successful fragmentation
• An OD quantitation of each resuspended sample
The Biomek FXP Target Prep Express can be set to prepare fragmentation and OD plates that are ready for processing. These process controls must be selected as a run preference prior to starting a run. See ʺSet the Biomek software default settingsʺ on page 25 for instructions.
1. Open Project Open Project Axiom 2.0 Target Prep and click OK.
2. Click the Open Method icon, select Axiom 2.0 Target Prep, and click OK.
3. Click the Run button.The Axiom 2.0 Method selection dialog box appears (Figure 39).
78 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 3: Resuspension and hybridization preparation 3
4. Select Resuspension and Hybridization Preparation and click OK.The deck layout for this method is displayed (Figure 40 on page 80).
5. Place the labware and reagents on the deck as directed in the following figures and table:
• Figure 40 on page 80—deck layout
• Figure 41 on page 81—location names of empty deck positions
• Table 20 on page 81—table detailing the labware, reagent and modular reservoir placement for the deck layout
• Figure 42 on page 83—reagent cold block
6. Label the Bio-Rad plates placed on the deck in positions P2 and P11. For example:
• P2—Hyb Ready + <sample description>
• P11—Gel QC
Note: Verify the appropriate plastic consumables are being used on the deck for the Hyb Reaction Plate when using the Applied Biosystems 9700 or Applied Biosystems 2720 thermal cycler.
Figure 39 Selecting the Resuspension and Hybridization Preparation method
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 79
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 3: Resuspension and hybridization preparation3
Figure 40 Deck layout for the Resuspension and Hybridization Preparation method
If Prepare plates for gel QC and OD after resuspension is selected in your run preferences, the following labware is required on the deck:
• Multichannel P50 pipette tips• Dilution QC plate• Gel QC plate• OD QC plate (do NOT touch bottom of
plate)If not selected, these plates will not appear in the deck layout.
See "Set the Biomek software default settings" on page 25 for more information.
80 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 3: Resuspension and hybridization preparation 3
Figure 41 Empty deck positions
Table 20 Labware and reagent locations on the deck for Resuspension and Hybridization Preparation
Position on deck
Labware Reagent or samples
TL1 Biomek AP96 – P250 Pipette Tips (aqua) —
P1 ABgene 96 Square Well Storage plate Pelleted samples
P2 Bio-Rad Hard-Shell 96-well plate(Hyb Reaction Plate).• Bio-Rad Hard-Shell 96-well plate (HSP-9631) for on-deck or
off-deck thermal cycling using the TRobot 96, PTC-200 or PTC 0240. OR
• Bio-Rad Hard-Shell Full-Height 96-Well Semi-Skirted PCR Plate (HSS-9601) stacked on a Costar Round Bottom plate for off-deck thermal cycling with the Applied Biosystems 9700 or 2720 thermal cycler
WARNING: When using the Applied Biosystems 9700 or the Applied Biosystems 2720 off-deck thermal cycler for denaturing the Hyb Ready Plate, the Bio-Rad HSS-9601 Hard-Shell Full-Height 96-Well Semi-Skirted PCR Plate must be stacked on a CoStar Brand Serocluster Round Bottom plate (29442-392 from VWR International or EK-680568 from E&K Scientific) as shown in Table 6 on page 29.The HSS-9601 and HSP-9631 PCR plates are not interchangeable on the Biomek FXP deck.
P3 Biomek Span P50 Pipette Tip (pink) —
P7 Bio-Rad Hard-Shell 96-well plate —
P8 Bio-Rad Hard-Shell 96-well plate
P9 Bio-Rad Hard-Shell 96-well plate
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 81
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 3: Resuspension and hybridization preparation3
P10 Reservoirs in frame:• Quarter module (1)• Quarter module (2)• Quarter module (3)• Quarter module, divided (4 and 5)
• Pour Axiom Resus Buffer into reservoir 1• Pour Axiom Hyb Buffer into reservoir 2• Leave reservoir 3 empty• Pour diluted gel loading buffer into reservoir 4: 13 mL• Pour nuclease-free water into reservoir 5 to fill line
P11 Bio-Rad Hard-Shell 96-well plate (for Gel QC) —
P12 OD plate, 96-well UV —
Pelt_1 Reagent block, chilled to 4°C See Figure 42 on page 83.
P13 Biomek Span P250 Pipette Tips (green) —
P14 Biomek Span P1000 Pipette Tips (yellow) —
Table 20 Labware and reagent locations on the deck for Resuspension and Hybridization Preparation
Position on deck
Labware Reagent or samples
1Axiom
ResuspBuffer
2AxiomHyb
Buffer
3Empty
Reservoir
4Gel Load
Buffer
5Water
82 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 3: Resuspension and hybridization preparation 3
7. Check the deck layout to ensure that the labware, reagents and samples are in the proper locations.
Note: If the physical deck does not match the Deck Layout window exactly (Figure 40 on page 80), either modify the physical deck to match exactly or choose Abort in the Deck Layout window.
8. Click OK to continue the method.
The system will automatically flush the Span-8 fluidics system. Observe the lines and syringes for air bubbles.
9. At the prompt to repeat the Span-8 fluidics system flush:
• Click No if no air bubbles are present.
• Click Yes if air bubbles are present. Repeat the flush until no air bubbles are present.
Figure 42 Placement of reagents on chilled reagent block for Resuspension and Hybridization Preparation
IMPORTANT:
• Position all plates and the chilled reagent block with A1 in the upper left corner of the frame.• Centrifuge all reagent tubes prior to placing in block to avoid loss of solution volume to the cap and sides of the tube.• Press reagent tubes into the block to ensure that they are fully seated.
Axiom Hyb
Soln 2
Axiom Hyb
Soln 1
Reagent block with template.
Only the positions used for this method are shown.
Diagram of reagent block without template
A1
A1
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 83
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 3: Resuspension and hybridization preparation3
10. Run the fragmentation gel and OD quantitation process controls.
For instructions and guidelines on assessing gel and OD results, see:
• Appendix B, ʺFragmentation quality control gel protocolʺ on page 292.
• Appendix C, ʺSample quantitation after resuspensionʺ on page 295.
11. Do one of the following:
• If the GeneTitan MC Instrument is free, and if the gel and OD quantitation results are good:
• Discard used labware and reagents.
• Discard used tips.
• Store unused Span-8 tips.
• Click OK in the Resuspension and Hyb Prep complete dialog box and proceed to ʺStage 4: Preparation for the GeneTitan™ MC Instrumentʺ on page 89.
• If the GeneTitan MC Instrument is not free, then follow the instructions shown in Figure 43:
• Tightly seal the Hyb Rxn plate and store at –20°C.
• Discard used labware and reagents.
• Discard used tips.
• Store unused Span-8 tips.
• Store cold block at 4°C.
• Click OK when complete.
Figure 43 Instructions to follow if the GeneTitan MC Instrument is not free
84 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 3: Resuspension and hybridization preparation 3
Summary of Resuspension and Hybridization Preparation
Stage 3: Resuspension & Hybridization Prep
Span-8MC
Stage 3: Resuspension & Hybridization Prep Page 1
Fill Resuspension Buffer Plate
ResB Reservoir ResB Plate
45 μL/well
Resuspend Pellet
ResB Plate Precip Plate
35 μL/well
Prepare Hybridization Master Mix & Distribution Plate
Hyb Soln 2 Hyb Soln 1
[96] 1197 μL [96] 66.5 μL
(4°C) (4°C)MM Reservoir
Hyb Plate
90 μL/well
Hyb Buffer
(RT)
[96] 9376.5 μL
Continued on ResuspensionPage 2
[96] FORMAT TRANSFER MIX MOVE
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 85
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 3: Resuspension and hybridization preparation3
Stage 3: Resuspension & Hybridization Prep
Span-8MC
Stage 3: Resuspension & Hybridization Prep Page 2
Continued from ResuspensionPage 1
Transfer Hybridization Master Mix to Precipitation Plate
Hyb Plate Precip Plate
80 μL/well
See QC flowchart if performing analysis after resuspension
Transfer Hybridization Mix to Hybridization Plate
Precip Plate Hyb Rxn Plate
115 μL/well
Store HybridizationReaction Plate
[96] FORMAT TRANSFER MIX MOVE
86 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 3: Resuspension and hybridization preparation 3
Stage 3: QC
Span-8MC
Stage 3: QC Page 1
Fill Dilution QC Plate with Water
Water Reservoir Dilution QC Plate
55 μL/well
Fill OD QC Plate with Water
Water Reservoir OD QC Plate
90 μL/well
Fill Gel QC Plate with Dye
Dye Reservoir Gel QC Plate
120 μL/well
Continued on QCPage 2
Transfer Hybridization Master Mix to Dilution Plate
Hyb Rxn Plate Dilution QC Plate
5 μL/well
[96] FORMAT TRANSFER MIX MOVE
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 87
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 3: Resuspension and hybridization preparation3
Perform Offline Analysis
Stage 3: Resuspension & Hybridization Prep
Span-8MC
Stage 3: QC Page 2
Transfer Diluted Sample to OD QC Plate
Dilution QC Plate OD QC Plate
10 μL/well
Continued from QCPage 1
Transfer Diluted Sample to Gel QC Plate
Dilution QC Plate Gel QC Plate
3 μL/well
[96] FORMAT TRANSFER MIX MOVE
88 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 4: Preparation for the GeneTitan™ MC Instrument 3
Stage 4: Preparation for the GeneTitan™ MC Instrument
About Stage 4 You will proceed to Stage 4 in 1 of 2 ways:
• Directly from Stage 3 without interruption.
• With samples that were stored at –20°C after Stage 3.
This stage, Preparation for GeneTitan, can include any combination of the options shown in Figure 44. The first 2 options complete target preparation on the Biomek FXP Target Prep Express.
The options are:
Option 1
• Denature samples—the Hyb Rxn plate is placed on the thermal cycler and the samples are denatured. At this step, you must also select Transfer denatured samples to hybridization tray. After the denature program has completed, the block will hold temperature until the user has dismissed the prompt, indicating that they are ready to continue the method to transfer the samples to the hybridization tray and then carry it to the GeneTitan MC Instrument. Do not leave samples on the thermal cycler for a long period of time.
Option 2
• Transfer denatured samples to hyb tray—the denatured samples are transferred from the Hyb Rxn plate to the hybridization tray, and are ready to load onto the GeneTitan MC Instrument.
Note: When using an Applied Biosystems 9700 or the Applied Biosystems 2720 thermal cycler for off-deck denaturation, the Bio-Rad Hard-Shell Full-Height 96-Well Semi-Skirted PCR Plate (Hyb Reaction Plate) must be stacked on a Costar brand Serocluster Round Bottom plate from Corning (Corning Mfg Cat. No. 3795).
Figure 44 Software setup for Preparation for GeneTitan
You can run one step, or a combination of steps.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 89
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 4: Preparation for the GeneTitan™ MC Instrument3
Option 3
• Prepare GeneTitan™ reagent trays - the solutions required for the fluidics stage of array processing on the GeneTitan MC Instrument are prepared and aliquoted to the appropriate trays (3 stain trays, 1 ligation tray, 1 stabilization tray and 1 scan tray with Holding Buffer).
When performing a 1 plate workflow, select 2 options (option 1 and option 2).
• Denature samples and Transfer denatured samples to hyb tray when you are preparing the samples to begin hybridization in the GeneTitan MC Instrument.
- or select 1 option (option 3)
• Prepare GeneTitan™ reagent trays to prepare reagents for loading onto the GeneTitan MC Instrument the following day when the plate is finished with the hybridization period and about to begin GeneTitan fluidics processing.
Options for performing a multi-plate workflow
When performing a multi-plate workflow (see Chapter 6, ʺProcessing 8 Axiom™ array plates per weekʺ on page 246 for a description of the 8 plate workflow), the need to carry out preparation of reagents for 1 plate while simultaneously hybridizing a second plate will arise. In this case, all 3 options in the Biomek FXP method can be carried out concurrently. Select all 3 options when using an on-deck thermal cycler.
• Denature samples and Transfer denatured samples to hyb tray will prepare samples for the new plate going into the GeneTitan MC Instrument to begin hybridization.
-and-
• Prepare GeneTitan™ reagent trays will prepare reagents for the plate that is already in the GeneTitan MC Instrument hybridization oven and is ready to go into the Wash, Ligation, Stain, and Scan phases of GeneTitan array processing.
Note: In the 8 plate workflow, all 3 options will only be selected for middle days of the workflow. On the first day of the workflow, only the Denature samples and Transfer denatured samples to hyb tray options will be used. On the last day of the workflow, only the Prepare GeneTitan reagent trays option will be used.
Note: When using an off-deck thermal cycler, avoid letting the samples sit a room temperature for an extended period of time after denaturation. Do not begin denaturation at the same time as the GeneTitan reagent preparation.
Off-deck thermal cycler option
The steps for performing a simultaneous preparation of GeneTitan reagent trays and hybridization of a second plate required in the course of a multi-plate workflow are modified slightly when the off-deck thermal cycler option is selected. The reagent trays to be loaded into the GeneTitan MC instrument are prepared in an initial run of the method.
Denaturation of the hybridization ready samples in the off-deck thermal cycler begins mid way through the GeneTitan reagent prep on the Biomek deck.
After loading the reagent trays into the GeneTitan MC instrument, you must perform a second run of the Biomek method to transfer the denatured samples to the hybridization tray for loading into the GeneTitan MC instrument. You will need a timer for this modified step.
90 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 4: Preparation for the GeneTitan™ MC Instrument 3
The modified steps are:
1. Select Preparation for GeneTitan step with only the Prepare GeneTitan™ reagent trays box checked, click OK.
2. Prepare deck as shown in Figure 45.
3. Click OK.
4. Start timer. Wait 25–30 minutes, then begin denaturation of the hybridization ready samples using the off-deck thermal cycler.
5. Upon completion of the GeneTitan reagent tray preparation, load reagent trays and scan tray into the GeneTitan MC instrument
6. Once the reagents are loaded into the GeneTitan MC instrument and the denaturation method on the thermal cycler is complete, retrieve the denatured hybridization ready samples from the thermal cycler
7. Return to Biomek FXP and begin a new method, select Preparation for GeneTitan step with only the Transfer denatured samples to hyb tray box checked, click OK.
8. Prepare deck as shown in Figure 46 (note that a spacer must be used with HSS-9601 plates for Applied Biosystems thermal cyclers).
Figure 45 Deck layout for off-deck thermal cycler option
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 91
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 4: Preparation for the GeneTitan™ MC Instrument3
9. Click OK.
10. After the denatured samples have been transferred to the GeneTitan hybridization tray, load hybridization tray into the GeneTitan MC Instrument.
Figure 46 Deck layout for off-deck thermal cycler option and position P2
-
Bio-Rad Hard-Shell Full-Height 96-Well Semi-Skirted
PCR Plate (HSS-9601) stacked on a Costar Round
Bottom plate for off-deck thermal cycling with the
Applied Biosystems 9700 or 2720 thermal cycler
The Hyb Reaction Plate in deck position P2 must be
one of the following:
Bio-Rad Hard-Shell 96-well plate (HSP-9631) for
on-deck or off-deck thermal cycling using the
TRobot 96, PTC-200 or PTC 0240
OR
--
92 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 4: Preparation for the GeneTitan™ MC Instrument 3
If a plate is already in the hybridization ovenFor the plate that is already in the GeneTitan hybridization oven and that is ready to go into Ligation, Wash-Stain and Scan, select option 3, Prepare GeneTitan™ reagent trays. The solutions required for the fluidics stage of array processing on the GeneTitan MC Instrument are prepared and aliquoted to the appropriate trays (3 stain trays, 1 ligation tray, 1 stabilization tray and 1 scan tray with Holding Buffer).
Duration of GeneTitan™ MC Instrument reagent preparation and sample preparation
Sample denaturation
Denatured sample transfer to hybridization tray
IMPORTANT! The reagent trays prepared in the third sub-step, Prepare GeneTitan™ reagent trays:
• Are NOT for use with the hybridization tray currently being prepared on the Biomek workstation.
• Are for the continued processing of an Axiom array plate that is:
– already on the GeneTitan MC Instrument.
– has completed the hybridization stage.
– is ready for transfer to the fluidics area.
The reagent trays for the fluidics stage on the GeneTitan MC Instrument CANNOT be prepared in advance. Do not prepare these plates if there is no array plate ready for the fluidics stage. Once prepared, these plates must be loaded onto the instrument as soon as possible and cannot be stored.
Table 21 Time required to denature samples
Activity Time
Hands-on time: ~3 minutes
Biomek FXP Target Prep Express 17 minutes
Total time: ~20 minutes
Table 22 Time required to transfer denatured samples to the hybridization tray
Activity Time
Hands-on time 2 minutes
Biomek FXP Target Prep Express ~2 minutes
Total time ~4 minutes
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 93
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 4: Preparation for the GeneTitan™ MC Instrument3
Preparation of GeneTitan reagent trays
Note: When you select Denaturation and Preparation of the GeneTitan Reagent Trays, the on-deck processes run concurrently.
Equipment and consumables required
Sample Denaturation
Transfer to hybridization tray
Table 23 Time required to prepare reagent trays for the GeneTitan MC Instrument
Activity Time
Prepare reagents (thaw and organize reagents)
~30 minutes
Hands-on time ~15 minutes
Biomek FXP Target Prep Express ~30 minutes
Total time ~75 minutes
Table 24 Consumable required for denaturing samples on-deck
Item Quantity
Lid, arched metal 1
Table 25 Consumables required for transferring denatured samples to a hybridization tray
Item Quantity
Item required from the GeneTitan™ Consumable Kit, Cat. No. 901606:• Hybridization Tray 1
Pipette tips, Biomek AP96 – P250 (aqua) 1 full box
If using off deck Applied Biosystems 9700 or Applied Biosystems 2720 thermal cyclers: Costar brand Serocluster Round Bottom plate from Corning Mfg. Cat. No. 3795) to be used for stacking under the HSS-9601 semi-skirted plate (Hyb Reaction Plate).
1
94 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 4: Preparation for the GeneTitan™ MC Instrument 3
Prepare GeneTitan™ Reagent Trays
Note: See Table 7 on page 36 for GeneTitan MC Instrument consumable catalog numbers.
1. Perform the pre-run checklist
The following actions are the same as described under ʺ1. Perform the pre-run checklistʺ on page 53. Refer back to this step for details. Some or all of these steps may not be required depending upon the current state of the Biomek workstation.
To perform the pre-run checklist:
1. Power on the Biomek FXP Target Prep Express and all peripherals.
2. Check the water and waste containers; replenish or empty as required.
3. Launch the Biomek Software.
4. If open, close the thermal cycler lid (on page 53).
5. Home all axes (on page 56).
Table 26 Consumables and other equipment required
Item Quantity
Items required from the GeneTitan™ Consumable Kit, Cat. No. 901606: • Scan tray (with cover and protective base)• Stain trays• Covers for stain trays
155
Pipette tips, Biomek AP96 – P250 (aqua) 1 full box
Pipette tips, Biomek Span P1000 (yellow) 1 full box
Pipette tips, Biomek Span P250 (green) 1 full box
Reagent block, chilled to 4°C 1
Reservoirs, quarter module divided 3
Reservoirs, quarter module 3
Tube rack, 24 position with insert (room temperature rack) 1
Zerostat Anti-Static Gun 1
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 95
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 4: Preparation for the GeneTitan™ MC Instrument3
2. Prepare the reagents for GeneTitan Reagent Tray Preparation
Note: Ligation Buffer and Ligation Solution 2 require approximately 30 to 40 minutes to thaw on the benchtop at room temperature.
Reagents required
Preparing Axiom Wash A and Axiom Stabilize DiluentDuring storage of the Axiom Wash A and Axiom Stabilize Diluent (in Module 4-2 stored at 4°C), precipitation in the form of clear crystals can sometimes occur. Therefore, follow the procedure below to ensure that any precipitate is returned to solution prior to use.
Note: The presence of some precipitate is OK and will not adversely impact assay performance. Follow the instructions below to resuspend any precipitate before use.
Table 27 Reagents required for GeneTitan MC Instrument Reagent Tray Preparation
Module Reagent Thaw on benchtop, then place on ice
Place on ice Place on benchtop at room temperature
Module 3Room
Temperature
Axiom Wash Buffer A
Axiom Wash Buffer B
Axiom Water
Module 4-1–20°C
Axiom Ligate Buffer - for 30 minutes
Axiom Ligate Enzyme Keep at –20°C until ready to use
Axiom Ligate Soln 1
Axiom Probe Mix 1
Axiom Stain Buffer
Axiom Stabilize Soln
Module 4-22 to 8°C
Axiom Ligate Soln 2 - for 30 to 40 minutes
Axiom Probe Mix 21
Axiom Wash A - for 30 minutes
Axiom Stain 1-A1
Axiom Stain 1-B1
Axiom Stain 2-A1
Axiom Stain 2-B1
Axiom Stabilize Diluent
Axiom Water
Axiom Hold Buffer1
1 These solutions are light sensitive. Keep tubes out of direct light for a prolonged period of time.
96 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 4: Preparation for the GeneTitan™ MC Instrument 3
To prepare the Axiom Wash A:
1. Vortex the bottle for 30 seconds.
2. Place on the benchtop at room temperature for 30 minutes.
3. Examine the reagent for precipitate (look into the top of the bottle).
4. If precipitate is still present, vortex again for 30 seconds.
5. Pour Axiom Wash A into the appropriate reagent reservoir and leave on the benchtop until ready to use.
To prepare the Stabilize Diluent:
If crystals are observed in the Axiom Stabilize Diluent:
1. Vortex and centrifuge.
2. Look for precipitate.
If any: Warm tube to room temperature and vortex again.
Preparing Axiom Ligate BufferWhite precipitate is sometimes observed when the Axiom Ligate Buffer is thawed.
Note: The presence of some precipitate is okay and will not adversely impact assay performance. Follow the instructions below to attempt to resuspend a majority of precipitate before use.
To prepare the Axiom Ligate Buffer:
1. Place on the benchtop at room temperature for 30 minutes. This bottle can also be thawed in a dish with room temperature Millipore water.
2. Examine the buffer for precipitate (look into the top of the bottle).
3. If precipitate is present, vortex the bottle for 30 seconds.
4. Re-examine the buffer for precipitate.
5. If precipitate is still present, warm the bottle with your hands and vortex again for 30 seconds.
6. If precipitate is still present after hand warming proceed with the protocol below.
7. Pour the Axiom Ligate Buffer into the appropriate reagent reservoir and leave on the benchtop until ready to use.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 97
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 4: Preparation for the GeneTitan™ MC Instrument3
Prepare the remaining reagents
1. Leave the Axiom Ligate Enzyme at –20°C until ready to use.
2. Thaw the following reagents from Module 4-1 at –20°C on the benchtop at room temperature, then vortex, centrifuge and place on ice:
• Axiom Ligate Soln 1
• Axiom Probe Mix 1
• Axiom Stabilize Soln
• Axiom Stain Buffer
3. Prepare the remaining reagents from Module 4-2 as follows:
a. Gently flick each tube 2 to 3 times to mix, then centrifuge.
b. Place reagents on ice, except for the Axiom Hold Buffer, Axiom Ligate Soln 2 and Axiom Water— leave these reagents on the benchtop at room temperature until ready to use.
3. Prepare the Sample Plate (if stored at –20°C) and the array plate
To prepare samples that were stored at –20°C:
1. Warm up the Hyb Ready Plate at room temperature for 5 minutes. It is not necessary to equilibrate the plate for longer duration.
2. Ensure that the Hyb Ready Plate is sealed well. If the plate is not sealed well:
a. Centrifuge the plate and carefully remove the old seal.
b. If there is condensation on the top of the plate, blot dry gently with a Kimwipes laboratory tissue.
c. Use a fresh seal and tightly reseal the plate.
3. Vortex the Hyb Ready Plate briefly, then centrifuge to 1,000 rpm.
4. Place the Hyb Ready Plate at room temperature until ready for use.
To prepare the array plate:
1. Warm up the array plate on the benchtop before setting up hybridization on the GeneTitan MC Instrument.
a. Leave the array plate in the pouch at room temperature, for a minimum of 25 minutes, before opening and loading on the GeneTitan MC Instrument to allow the plate to come to room temperature.
b. At the end of the array warm up time, open the pouch and scan the array plate barcode into the Batch Registration file (see ̋ Stage 1: Create and upload Batch Registration fileʺ on page 219).
WARNING! Do not remove the array plate from the protective base or touch the surface of any arrays.
98 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 4: Preparation for the GeneTitan™ MC Instrument 3
4. Prepare the GeneTitan™ MC Instrument
Before you denature your hybridization ready samples, ensure that the GeneTitan MC Instrument is ready for use.
One or more of the following steps may need to be performed:
1. Launch Applied Biosystems™ GeneChip™ Command Console and select GCC GeneTitan Control.
2. Upload your sample registration file now, if you have not done so (see ʺStage 1: Create and upload Batch Registration fileʺ on page 219).
If you do not upload your samples before scanning the array plate barcode, the software will assign names to your samples.
3. On the GCC GeneTitan Instrument Control, select the System Setup tab (Figure 47).
4. Configure the software as follows:
a. Setup Option: Hyb-Wash-Scan
Other options available are described under ʺSetup options for array plate processingʺ on page 215.
b. Click Next.
c. Plate Information:
• Barcode: Scan or manually enter the array plate barcode and click Next.
• Protocol Name: Select the protocol name and click Next.
5. Fill the Wash A, Wash B and Rinse bottles.
6. Empty the Waste bottle.
After preparing the GeneTitan MC Instrument, perform steps 6a, 6b or 6c as appropriate for your workflow:
• Go to Step 6a on page 106: if you are denaturing the samples and transferring them to the hybridization tray.
• Go to Step 6b on page 107: if you are preparing reagents for the Ligation-Wash-Stain.
• Go To Step 6c on page 107: if you are performing a 2-8 plate workflow, and you are denaturing samples for the 2nd plate and are preparing ligation reagents for the 1st plate. The samples for the 2nd Axiom array plate will be denatured and transferred into the hybridization tray. The hybridization tray will be placed in the GeneTitan MC Instrument with the array plate in preparation for
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 99
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 4: Preparation for the GeneTitan™ MC Instrument3
hybridization. The Biomek FXP. Target Prep Express will also prepare reagents for Ligation-Wash-Stain for the first array plate that was placed in the GeneTitan MC Instrument hybridization oven 24 hours ago.
5. Run the Preparation for GeneTitan™ step
To run the Preparation for GeneTitan step:
1. Open Project Open Project Axiom 2.0 Target Prep and click OK.
2. Click the Open Method icon, select Axiom 2.0 Target Prep, and click OK.
3. Click the Run button.The Axiom 2.0 Target Prep run options window appears (Figure 48).
4. Select Preparation for GeneTitan™ and the sub-steps that you wish to run; then click OK.
Based upon your choices, the appropriate deck layout is displayed (Figure 49).
Figure 47 Setup Options for processing array plates
System Setup tab
Select Hyb-Wash-Scan option
Figure 48 Software setup for Preparation for GeneTitan
You can run each step individually, or any combination of steps. If you do not select Prepare GeneTitan™ reagent trays, the deck layout will be different than the one shown in Figure 49.
• Select Denature Samples and Transfer denatured samples to hybridization tray– if you are preparing to load the array plate into the GeneTitan
Instrument for Hybridization.• Select Prepare GeneTitan™ Reagent Trays
– if you are preparing to load the GeneTitan Instrument for Wash-Stain and Imaging.
• Select Denature Samples and Transfer denatured samples to hyb tray and Prepare GeneTitan™ Reagent trays
– if you are running an 8-plate workflow then you will need to select all 3 check boxes in some instances in preparation for the plate that will go into the GeneTitan Instrument for Hybridization.
• Select Prepare GeneTitan™ Reagent trays for the array plate that has completed hybridization and is ready to be processed in the GeneTitan Instrument for Ligation- Wash-Stain and Imaging.
100 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 4: Preparation for the GeneTitan™ MC Instrument 3
5. Deionize the GeneTitan stain trays. See Appendix E, section entitled ʺDeionization procedure for GeneTitan™ trays and coversʺ on page 307 for the deionization procedure.
6. Place the labware and reagents on the deck as directed in the following figures and table:
• Figure 49 on page 101—deck layout
• Figure 50 on page 102—location names of empty deck positions
• Table 28 on page 102—table detailing the labware, reagent and modular reservoir placement for the deck layout
• Figure 51 on page 104—reagent cold block
• Figure 52 on page 105—tube block
Note: Prior to placing the arched metal lid on the deck, be sure to clean the attached Microseal® P pad with 70% ethanol and dry it. See the product information sheet for this product for further information on cleaning and replacement.
IMPORTANT! It is important to deionize the GeneTitan MC Instrument trays to remove any static electricity on the trays. Static attraction by the trays may prevent the tray cover from being lifted up by the instrument.
Figure 49 Deck layout for Preparation for GeneTitan—denature, transfer to hybridization tray, and GeneTitan plate preparation.
The deck layout displayed is based upon the selections you make for this step.
IMPORTANT: Destatic the stain trays prior to placing them on the deck. See the section "Deionization procedure for GeneTitan™ trays and covers" on page 307.
Included in deck layout if:
• Denature samples• Transfer denatured samples to
Hyb Tray are selected.
Included in deck layout if Prepare GeneTitan™ reagent trays is selected.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 101
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 4: Preparation for the GeneTitan™ MC Instrument3
Figure 50 Empty deck positions
Table 28 Labware and reagent locations on the deck for GeneTitan Reagent Preparation
Position on deck
Labware Reagent or samples
If Denature samples and Transfer denatured samples to Hyb Tray is selected, (no reagent tray preparation), only the labware listed below is required:
TL1 Biomek AP96 – P250 Pipette Tips (aqua) —
P1 Lid, arched metalClean before use (70% ethanol).
—
P2 Hyb Reaction Plate1 Hyb-ready samples
P3 Hybridization tray2 —
If Prepare GeneTitan™ reagent trays, the labware listed below is required:
P4 Stain 12, 3 (second of 2 trays with Stain 1) —
P5 Stain 22, 3 —
P6 Lig2, 3 —
P7 Scan Tray on protective base* —
P8 Stbl2, 3 —
P9 Stain 12, 3 (first of 2 trays with Stain 1) —
P10 Tube block with 1 insert, room temperature See Figure 52 on page 105.
P11 Reservoirs in frame:• Quarter module, divided (1 and 2)• Quarter module, divided (3 and 4)• Quarter module, divided (5 and 6)
• Pour Axiom Water into reservoir 1• Pour Axiom Ligation Buffer into reservoir 5
1Axiom Water
3Empty
5Axiom LigateBuffer
2Empty
4Empty
6Empty
102 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 4: Preparation for the GeneTitan™ MC Instrument 3
Labeling GeneTitan hybridization and reagent traysWhen preparing the hybridization and reagent trays to be loaded onto the GeneTitan MC Instrument, you will need to mark each tray in a way that identifies its contents.
P12 Reservoirs in frame:• Quarter module (1)• Quarter module (2)• Quarter module (3)
• Pour Axiom Hold Buffer into reservoir 1• Pour Axiom Wash A into reservoir 2• Leave reservoir 3 empty
Pelt_1 Reagent block, chilled to 4°C See Figure 51 on page 104
P13 Biomek Span P250 Pipette Tips (green) —
P14 Biomek Span P1000 Pipette Tips (yellow) —
1 Bio-Rad Hard-Shell 96-well plate (HSP-9631) for on-deck or off-deck thermal cycling using the TRobot 96, PTC-200 or PTC 0240. Bio-Rad Hard-Shell Full-Height 96-Well Semi-Skirted PCR Plate (HSS-9601) stacked on a Costar Round Bottom plate for off-deck thermalcycling with the Applied Biosystems 9700 or 2720 thermal cycler.
2 These trays are included in Axiom Genome-Wide and Custom myDesign™ Array Plate Kits.3 Label each of these stain trays as described above as described in "Labeling GeneTitan hybridization and reagent trays". For example,
label the Stain tray to be placed in P6 with the word Lig. This tray will contain the Ligation master mix.
Table 28 Labware and reagent locations on the deck for GeneTitan Reagent Preparation (Continued)
Position on deck
Labware Reagent or samples
1Axiom Hold
Buffer
2Axiom
Wash A
3Empty
Reservoir
IMPORTANT! It is critical that you write only on the proper location of the hybridization tray (on the edge in front of wells A1 and B1) as illustrated in Figure 13 on page 44 or on the proper location of the stain/reagent trays (on the edge in front of wells A1 to C1) as illustrated in Figure 14 on page 45. Do NOT write on any other side, as this can interfere with sensors inside of the GeneTitan MC Instrument and result in experiment failure. To ensure proper placement of lids onto stain trays, and trays onto the GeneTitan MC Instrument, you can also mark the notched corner of the trays and lids.
IMPORTANT! Do not confuse hybridization trays with stain trays.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 103
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 4: Preparation for the GeneTitan™ MC Instrument3
Figure 51 Placement of reagents on chilled reagent block for GeneTitan Reagent Tray Preparation.
IMPORTANT:
• Position all plates and the chilled reagent block with A1 in the upper left corner of the frame.• Centrifuge all reagent tubes prior to placing in block to avoid loss of solution volume to the cap and sides of the tube.• Press reagent tubes into the block to ensure that they are fully seated.
Axiom Ligate
Enzyme
Axiom Probe Mix 1
Axiom Probe Mix 2
Axiom Ligate Soln 1
Axiom Stain 2-B
Axiom Stain Buffer
Axiom Stabilize
Soln
Axiom Stabilize Diluent
Axiom Stain 2-A
Axiom Stain 1-B
Axiom Stain 1-A
Reagent block with template.
Only the positions used for this method are shown.
Diagram of reagent block without template
A1
A1
104 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 4: Preparation for the GeneTitan™ MC Instrument 3
7. Check the deck layout to ensure that the labware, reagents and samples are in the proper locations.
Note: If the physical deck does not match the Deck Layout window exactly (Figure 49 on page 101), either modify the physical deck to match exactly or choose Abort in the Deck Layout window.
8. Click OK to continue the step.
The system will automatically flush the Span-8 fluidics system. Observe the lines and syringes for air bubbles.
9. At the prompt to repeat the Span-8 fluidics system flush:
• Click No if no air bubbles are present.
Click Yes if air bubbles are present. Repeat the flush until no air bubbles are present.
If the “Denature Samples” option is selected, the Biomek FXP Target Prep Express places the samples onto the thermal cycler and runs the denaturation program. If Prepare GeneTitan™ reagent trays is also selected, reagent tray preparation for the GeneTitan MC Instrument starts while the samples are being denatured.
Figure 52 Placement of reagents on room temperature tube block.
AxiomLigationSoln 2
A1
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 105
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 4: Preparation for the GeneTitan™ MC Instrument3
6a. Complete Stage 4: Preparation for GeneTitan™ - Hybridization Trays
If you selected Denature samples and Transfer denatured samples to Hyb tray in Step 4 (See Step ʺ5. Run the Preparation for GeneTitan™ stepʺ on page 100 and Figure 48 on page 100), use the following instructions:
1. Once the GeneTitan MC Instrument is ready, return to the Biomek FXP Target Prep Express click OK (prompt shown in Figure 53 above).
The denatured samples are then taken off the thermal cycler and are transferred to the hybridization tray.
• Ensure that there are no air bubbles present in the hybridization tray. Puncture any air bubbles that you see using a pipette tip.
2. Transfer the hybridization tray to the GeneTitan MC Instrument and load. See the section ʺArray processing with the GeneTitan™ MC Instrumentʺ on page 209 for the proper way of loading the array plate and the hybridization tray.
3. Return to the Biomek FXP Target Prep Express and clear the deck (Figure 54).
• Always discard the used multichannel pipette tips in position P9.
• Always store the reagent block at 4°C.
• Clean the Microseal P Pad by wiping with 70% EtOH and dry.
See the product information sheet for this product for further information on cleaning and replacement.
Figure 53 Prompt indicating denaturation is complete.
IMPORTANT! Immediately load the array plate and hybridization tray into the GeneTitan MC Instrument.
Figure 54 Instructions displayed at the end of Step 4.
106 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 4: Preparation for the GeneTitan™ MC Instrument 3
6b. Complete Stage 4: Preparation for GeneTitan™ - Reagent Trays
If you selected Prepare GeneTitan™ Reagent trays in Step 4 (See Step ʺ5. Run the Preparation for GeneTitan™ stepʺ on page 100 and Figure 48 on page 100), use the following instructions:
1. Once the prompt shown in Figure 54 appears, remove the reagent trays and scan tray with Hold Buffer from the deck and cover with the appropriate lids.
2. Examine each tray to ensure that:
• All of the wells have been filled. If any wells do not contain reagents, then manually add reagents to these wells.
• There are no air bubbles present. Puncture any air bubbles that you see using a pipette tip.
j
3. Transfer the reagent trays, scan tray to the GeneTitan MC Instrument and load. See the section ʺStage 3: Ligate, Wash, Stain, and Scanʺ on page 235 to continue the process on the GeneTitan MC instrument.
4. Return to the Biomek FXP Target Prep Express and clear the deck (See Figure 47 on page 100).
• Always discard the used multichannel pipette tips in position P9.
• Always store the reagent block at 4°C.
• Clean the Microseal P Pad by wiping with 70% EtOH and dry.
See the package insert for this product for further information on cleaning and replacement.
6c. Complete Stage 4: Preparation for GeneTitan™ - multiple plate workflow
If you selected all 3 options in Step 4 (see Step ʺ5. Run the Preparation for GeneTitan™ stepʺ on page 100 and Figure 48 on page 100) and are preparing hybridization tray and reagent trays in a 2+ plate workflow, use the following instructions:
The hybridization tray is prepared for a new Axiom array plate that will be loaded into the GeneTitan MC Instrument. The reagent trays are prepared for the Axiom array plate that is in the hybridization oven in the GeneTitan MC Instrument and is ready to move to the next stage of the process - Ligation, Wash-Stain and Scan.
Note: When using an off-deck thermal cycler see the instructions for the ʺOff-deck thermal cycler optionʺ on page 90.
1. Once the reagent trays are prepared and sample denaturation is complete, the prompt in Figure 53 on page 106 is displayed (do not click OK yet). Remove the reagent trays and scan tray with Hold Buffer from the deck and cover with the appropriate lids.
2. Examine each tray to ensure that:
• All of the wells have been filled. If any wells do not contain reagents, then manually add reagents to these wells.
• There are no air bubbles present. Puncture any air bubbles that you see using a pipette tip.
IMPORTANT! Immediately load the reagent trays onto the GeneTitan MC Instrument. Do not leave denatured samples or reagent trays at room temperature for any length of time.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 107
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 4: Preparation for the GeneTitan™ MC Instrument3
3. Transfer the reagent trays, scan tray and array plate to the GeneTitan MC Instrument and load.
4. Return to the Biomek FXP Target Prep Express click OK at the prompt shown in Figure 53 on page 106.
The denatured samples are then taken off the thermal cycler and are transferred to the hybridization tray.
5. Transfer the hybridization tray to the GeneTitan MC Instrument and load. See the section ʺArray processing with the GeneTitan™ MC Instrumentʺ on page 209 for the proper way of loading.
6. Transfer the reagent trays, scan tray to the GeneTitan MC Instrument and load. See the section ʺArray processing with the GeneTitan™ MC Instrumentʺ on page 209 to continue the process on the GeneTitan MC Instrument.
7. Return to the Biomek FXP Target Prep Express and clear the deck (Figure 54 on page 106).
• Always discard the used multichannel pipette tips in position P9.
• Always store the reagent block at 4°C.
• Clean the Microseal® P Pad by wiping with 70% EtOH and dry.
See the package insert for this product for further information on cleaning and replacement.
IMPORTANT! Immediately load the reagent trays and the hybridization tray onto the GeneTitan MC Instrument. Then load the array plate and hybridization tray. Do not leave denatured samples or reagent trays at room temperature for any length of time.
108 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 4: Preparation for the GeneTitan™ MC Instrument 3
Summary of Preparation for GeneTitan™ MC Instrument
Stage 4: Preparation for the GeneTitan Instrument
Span-8MC
Stage 4: Preparation for the GeneTitan Instrument Page 1
Move Hyb Lid onto Hyb Rxn Plate
Hyb Lid Hyb Rxn Plate
Fill Scan Tray with Hold Buffer
Hold Buffer Reservoir Hold Buffer Plate
150 μL/well
Continued onGeneTitan Preparation - Page 2
[96] FORMAT TRANSFER MIX MOVE
Prepare Stabilize Solution & Plate
Water Stabi lize Soln
[96] 10727 μL [96] 144.96 μL
(RT) (4°C)Stabi lize Solution Reservoir
Stabi lize Solution Plate
105 μL/well
[ LLLL
Stabi lize Diluent
(4°C)
[96] 1208 μL
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 109
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 4: Preparation for the GeneTitan™ MC Instrument3
Stage 4: Preparation for the GeneTitan Instrument
Span-8MC
Stage 4: Preparation for the GeneTitan Instrument Page 2
Continued onGeneTitan Preparation - Page 3
Transfer Wash A Buffer to Stain 1 & 2 Reservoirs
Stain 1 Reservoir Stain 2 Reservoir
(RT)
Wash A Buffer
[96] 22233.6 μL [96] 11596.8 μL
Continued fromGeneTitan Preparation - Page 1
[96] FORMAT TRANSFER MIX MOVE
Transfer Stain Buffer to Stain 1 & 2 Reservoirs
Stain 1 Reservoir Stain 2 Reservoir
(4oC)
Stain Buffer
[96] 463.2 μL [96] 241.6 μL
Transfer Stains 1-A & 1-B to Stain 1 ReservoirStain 1-A Stain 1-B
[96] 231.6 μL [96] 231.6 μL
(4°C) (4°C)Stain 1 Reservoir
110 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 4: Preparation for the GeneTitan™ MC Instrument 3
Stage 4: Preparation for the GeneTitan Instrument
Span-8MC
Stage 4: Preparation for the GeneTitan Instrument Page 3
Continued onGeneTitan Preparation - Page 4
Continued from GeneTitanPage 2
Transfer Stains 2-A & 2-B to Stain 2 Reservoir
Stain 2-A Stain 2-B
[96] 120.8 μL [96] 120.8 μL
(4°C) (4°C)Stain 2 Reservoir
[96] FORMAT TRANSFER MIX MOVE
Incubate Hybridization Plate in PTC200
Hyb Rxn Plate + Lid PTC200
Incubate:
1) 10 mins @ 95°C
2) 3 mins @ 48oC
Prepare Stain 1 Plates
Stain 1Plate 1 Stain 1 Plate 2
Stain 1 Reservoir
105 μL/well 105 μL/well
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 111
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 4: Preparation for the GeneTitan™ MC Instrument3
Stage 4: Preparation for the GeneTitan Instrument
Span-8MC
Prepare Stain 2 PlateStain 2 Reservoir Stain 2 Plate
105 μL/well
Stage 4: Preparation for the GeneTitan Instrument Page 4
Continued onGeneTitan Preparation - Page 5
Continued fromGeneTitan Preparation - Page 3
Prepare Ligate Solution & Distribution Plate
Ligate Buffer Ligate Enzyme
[96] 7610.4 μL [96] 181.2 μL
(RT) (4°C)MM Reservoir
Ligate Plate
105 μL/well
Ligate Soln 1
(4°C) [96] 1510 μL
Ligate Soln 2
[96] 362.4 μL (4°C)
[96] 1208 μL [96] 1208 μL (4°C)(4°C)
Probe Mix 1 Probe Mix 2
L
[96] FORMAT TRANSFER MIX MOVE
112 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 3 Target preparation on the Biomek FXP with Windows® XPStage 4: Preparation for the GeneTitan™ MC Instrument 3
Stage 4: Preparation for the GeneTitan Instrument
Span-8MC
Stage 4: Preparation for the GeneTitan Instrument Page 5
Unload Hybridization Plate from PTC200
Hyb Rxn Plate + Lid Position P2
Remove Lid from Hybridization Plate
Position P2 Position P1
Process Hybridization Trayon the GeneTitan Instrument
Transfer Samples to Hybridization Tray
Hyb Rxn Plate Hyb Tray
105 μL/well
Continued fromGeneTitan Preparation - Page 4
[96] FORMAT TRANSFER MIX MOVE
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 113
4 Target preparation on the BiomekFXP with Windows® 7
This chapter is intended for users running the Axiom 2.0 automated target preparation method using the Biomek FXP Target Prep Express software on Windows 7. If you are currently using Windows XP, see Chapter 3, ʺTarget preparation on the Biomek FXP with Windows® XPʺ on page 22
Whatʹs new for the Axiom™ 2.0 Target Preparation 96-Samples Biomek® FXP Method for Windows® 7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
Axiom™ 2.0 Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Before using the Biomek workstation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Stage 1: DNA Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Stage 2: Fragmentation and Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Stage 3: Centrifugation and drying pellets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Stage 4: Resuspension, Hybridization Preparation, and QC . . . . . . . . . . . . . . . 171
Stage 5: Preparation for the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . 184
What's new for the Axiom™ 2.0 Target Preparation 96-Samples Biomek® FXP Method for Windows® 7
Several changes have been made to the Axiom 2.0 Target Preparation 96-Samples Method for Windows® 7. Follow the new deck layouts and instructions as displayed in the user interface (while running the method), in this user guide, or in the Axiom 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP (Windows 7) Quick Reference (Pub. No. 703369).
New features • Updated user interface:
– Improved deck setup screen, featuring images of actual labware.
– Clearly designated reservoirs and labware positions.
• Countdown timers have been implemented for all on-deck incubation steps to help users track the assay run.
• New Labware: The Precipitation Plate for the new Windows 7 method uses the Eppendorf deep well plate (Cat. No. 951033481). This plate replaces the ABgene square well storage plate (Thermo Scientific AB-0932) used in the Windows® XP method.
114 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Axiom™ 2.0 Assay 4
Method changes • Fragmentation method:
– The Fragmentation Master Mix now combines the Axiom 10X Frag Buffer, Axiom Frag Diluent, and Frag Enzyme into one master mix.
– The Isopropanol will now be added to the Precipitation Master Mix in the Precip Plate prior to the transfer of samples.
• Resuspension method:
– DNA pellet resuspension will now be performed off-deck using a microplate shaker.
– There will be 2 prompts guiding the users when to: 1) take the Sample Plate off the deck for off-deck pellet resuspension, and 2) when to return the plate back on the deck to finish the Resuspension method.
New off-deck step The user intervention steps during Resuspension now instructs the user to perform an off-deck DNA pellet resuspension step after the addition of the Axiom Resuspension Buffer. This 10-minute off-deck plate shaking protocol on a microplate shaker resuspends the pelleted DNA into the Axiom Resuspension Buffer.
Others • Updated Reservoir Stickers (Part No. 101135): The new Axiom™ 2.0 Assay method on the Biomek™ FXP Target Prep Express instrument with Windows® 7 requires new stick-on labels for the modular reagent reservoir frames for the different stages of the workflow. These stick-on labels have been updated to reflect the changes made in the Fragmentation method. They are color-coded to match the colors found on the caps of the reagent bottles in the Axiom 2.0 reagent kit.
• Labware Consumables Kit: Consumables kit for on-deck (TRobot) and off-deck (Applied Biosystems or PTC) thermal cycler users are now available to order through Thermo Fisher Scientific. See Table 34 ʺGuidance for consumable kit orderingʺ on page 124 to determine which consumables kits are applicable for your needs.
Axiom™ 2.0 Assay
The Axiom™ 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP (Windows 7) is designed for processing 96 samples at a time. The protocol is performed in 2 parts:
• Part 1: Target Preparation is performed on the Biomek FXP Target Prep Express
• Part 2: Array Processing is performed on the GeneTitan™ Multi-Channel (MC) Instrument
A list of all equipment and resources for the Axiom 2.0 Assay with Automated Target Prep is in the Axiom™ 2.0 Assay Automated Workflow for Windows® 7 Site Preparation Guide, Pub. No. 703368.
This chapter includes instructions for Part 1: Target Preparation. These instructions are presented as follows:
• ʺBefore using the Biomek workstationʺ on page 117
– ʺSeal, vortex and centrifugeʺ on page 117
– ʺBreaking the light curtainʺ on page 118
– ʺPlate centrifugeʺ on page 119
– ʺLabeling GeneTitan hybridization and reagent traysʺ on page 137
– ʺPipette tip usageʺ on page 118
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 115
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Axiom™ 2.0 Assay4
– ʺSetting method preferencesʺ on page 119
– ʺEquipment, consumables, labware, and reagentsʺ on page 123
– ʺReagent block templateʺ on page 142
– ʺRelated Biomek FXP Target Prep Express documentationʺ on page 144
• ʺStage 1: DNA Amplificationʺ on page 145
• ʺStage 2: Fragmentation and Purificationʺ on page 157
• ʺStage 3: Centrifugation and drying pelletsʺ on page 169
• ʺStage 4: Resuspension, Hybridization Preparation, and QCʺ on page 171
• ʺStage 5: Preparation for the GeneTitan™ MC Instrumentʺ on page 184
IMPORTANT! Before proceeding to DNA Amplification, do the gDNA preparation described in Chapter 2, ʺGenomic DNA preparation and requirementsʺ on page 13.
116 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Before using the Biomek workstation 4
Before using the Biomek workstation
Seal, vortex and centrifuge
Unless otherwise stated in the protocol, follow the guidelines below when instructed to seal, vortex and centrifuge plates or reagent tubes for the Biomek FXP Target Prep Express portion of this assay:
• Seal plates—we recommend using MicroAmp Clear Adhesive Films to seal your plates.
• Blot-dry: Prior to sealing plates, we recommend checking the top of the plate to ensure that there are no droplets. If droplets are present, blot-dry the top of the plate before sealing to ensure a tight seal.
– To remove droplets prior to sealing, overlay a sheet of Kimwipes laboratory tissue across the top of the plate and gently pat down to dry.
– Lift the sheet off the plate and discard. Confirm the top of the plate is dry and seal the plate as usual.
• Vortex:
– Reagents 3 times, 1 second each time at the maximum setting.
– Plates 1 second each corner, and 1 second in the center at the maximum setting.
• Centrifuge: When instructed to centrifuge plates or reagent vials, follow these guidelines unless otherwise instructed (for example, when centrifuging and drying pellets, ʺStage 3: Centrifugation and drying pelletsʺ on page 169).
– Plates:
• Centrifuge at 1,000 rpm for 1 minute at room temperature.
• Do not centrifuge for more than 1 minute.
– Reagent Vials: 3 seconds
IMPORTANT! Always ensure that your plates are tightly sealed. A tight seal will prevent sample loss and cross-well contamination, particularly when plates are being vortexed. NEVER REUSE A SEAL. ALWAYS USE A NEW SEAL.
Figure 55 Vortex plates at the corners and center
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 117
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Before using the Biomek workstation4
Breaking the light curtain
For your safety, the Biomek FXP Target Prep Express is designed to immediately halt all movement when the light curtain is broken.
Light curtain broken while running the assayFor your safety, the Biomek FXP Target Prep Express is equipped with a light curtain (Figure 56). The light curtain senses when an object (such as a hand or an arm) enters the space surrounding the deck. When this curtain is broken, all movement on the deck halts until the user either clicks OK to resume the activity that was taking place, or aborts the activity. Incubation timers are not interrupted.
Pipette tip usage
Figure 56 Prompt displayed when the light curtain is broken during the process referred to as Homing All Axes.
The light curtain covers the front of the instrument. The sides are protected by Plexiglas. Both areas are shown as red in this figure. Any penetration of the light curtain from outside the deck halts all movement of the workstation. To resume activity on the deck, click OK. To abort the step or other activity, click Stop on the toolbar.
Table 29 Pipette tip usage for 1 full run of the Axiom™ 2.0 Assay on the Biomek workstation
StepMulti-Channel
P50, Pink96 tips, Cat. No. A21586
Multi-Channel AP96 P250,
Aqua96 tips,
Cat. No. 717253
Span-8 Span P250,
Green96 tips,
Cat. No. 379503
Span-8 Span P1000,
Yellow96 tips,
Cat. No. 987925
96 rxns 96 rxns 96 rxns 96 rxns
DNA Amplification — 96 tips — 33 tips
Fragmentation — 96 tips 8 tips 36 tips
Resuspension and Hybridization Preparation 96 tips 96 tips 17 tips 23 tips
Preparation for the GeneTitan™ MC Instrument• Denature samples• Transfer denatured samples to hyb tray• Prepare GeneTitan™ reagent trays
— 96 tips 26 tips 96 tips
TOTAL 96 tips 384 tips 51 tips 188 tips
118 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Before using the Biomek workstation 4
Plate centrifuge One plate centrifuge is required for the Axiom™ 2.0 Assay. See the Assay 96-Array Format Automated Workflow for Biomek FXP Site Preparation Guide, Pub. No. 702984, for an appropriate plate centrifuge that can be used with the Axiom Genotyping Solution. When centrifuging and drying pellets as instructed under ̋ Stage 3: Centrifugation and drying pelletsʺ on page 169, the centrifuge must be able to spin down plates at:
• rcf: 3,200 x g (4,000 rpm for the Eppendorf 5810R with the rotor configuration described in the Axiom™ 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP Site Preparation Guide, Pub. No. 702984)
• Temperature: 4°C
In addition, the bottom of the rotor buckets should be soft rubber to ensure that the deep well plates do not crack. Do not centrifuge plates in metal or hard plastic buckets.
Setting method preferences
Typically you will set the method preferences for the Biomek software once. The settings you select will:
• Determine which process controls will be run during stages 2 and 3 (Fragmentation and Resuspension). The process controls include:
– Highly recommended: Preparation of sample dilution plates for OD and gel analysis during Resuspension and Hyb Prep. The dilution plates are taken off-deck. One is used for OD quantitation to evaluate DNA mass; the other is used to check fragment size.
– If desired: Prompt you to manually take samples for DNA quantitation prior to fragmentation.
• Select deck configuration options for the thermal cycler (choose between having an integrated Biometra TRobot 96 thermal cycler or no integrated thermal cycler).
To set the method preferences:
1. Launch the Biomek Software.
2. Open Project Open Project Axiom 2.0 Target Prep and click OK (Figure 57).
3. Open File Open to display the Open Method window (Figure 58).
Or click the Open Method icon
4. Select Axiom 2.0 Target Prep and click OK.
Figure 57 Opening the Axiom 2.0 Target Prep Project
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 119
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Before using the Biomek workstation4
.
5. In the left pane of the window, select Axiom Target Prep (Figure 59).
The method preferences selection window is displayed on the right.
Figure 58 Opening the Axiom Target Preparation method
The Axiom 2.0 Target Prep project folder must be displayed in this menu.
Figure 59 Click Startup Dialog to open the Method Preferences Selection window
The selections made in this box, Device Configuration Option, are displayed each time this window appears.
The options in the Custom Run Options box must be selected prior to starting a run.
You are not prompted to select any preferences at start up.
120 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Before using the Biomek workstation 4
6. Set Method Preferences:
Device Configuration Option— these settings can be changed at the start of each step
• What type of thermal cycler setup do you have?
• TRobot-select this option to perform the denaturation of the Axiom Hyb Ready Plate on the integrated Biometra TRobot 96 thermal cycler
• No integrated thermal cycler - select this option to perform the denaturation of the Axiom Hyb Ready Plate on an off-deck thermal cycler or if your Biomek does not have an integrated thermal cycler. A list of thermal cyclers that have been verified with the assay can be found below. When selecting this option, select the appropriate plate type that should be used for the Hyb Ready Plate.
– Select the Bio-Rad Skirted option when using the HSP-9631 plate for the PTC-200 or the Bio-Rad Tetrad 0240G thermal cycler.
– Select the Bio-Rad Semi-Skirted on Costar Round U-Bottom option for the Applied Biosystems 9700 or the Applied Biosystems 2720 thermal cycler. The Bio-Rad HSS-9601 plate must be stacked onto the Costar Round Bottom plate from Corning (VWR International Cat. No. 29442-392, E&K Scientific: EK 680568, Corning Mfg. Cat. No. 3795) for the Biomek FXP Target Prep Express to prepare the Hyb Ready Plate.
We have verified the performance of this assay using the Biometra TRobot 96 on the Beckman Biomek Target Prep Express liquid handler. We have also verified the performance of this assay using the following off-deck thermal cyclers, with 96-well blocks:
• Bio-Rad PTC-200G
• Applied Biosystems 9700 with a gold, silver or aluminum block
• Applied Biosystems 2720
• Bio-Rad / MJ Tetrad® 2 PTC-0240G
The performance of this assay has not been verified with other thermal cyclers.
Use of other thermal cyclers may result in assay failure and may violate the Axiom Array and Reagent replacement policy.
The thermocycler needs to be programmed with the “Axiom Denature” protocol:
a. 95°C 10 minutes
b. 48°C 3 minutes
c. 48°C hold
Use the heated lid option when setting up or running the protocol.
WARNING! Evaporation during denaturation can negatively impact assay performance. Use the recommended thermal cycler consumables and sealing film to eliminate condensation and evaporation. The arched, auto-sealing metal plate with P pads as shown in Table 35 on page 124 should be replaced as per the manufacturer’s recommendation. If using the TRobot, complete the lid setting per the manufacturerʹs instructions.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 121
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Before using the Biomek workstation4
Custom Run Options— These settings are displayed in this window only. You must select/deselect here.
• QC check points
• Prompt for manual DNA quantitation before fragmentation—the workstation will pause following inactivation of the DNA amplification reaction to allow you to manually remove an aliquot of each sample for off-line (manual) DNA quantitation. This extra quality control step is available for troubleshooting the DNA amplification reaction.
• Prepare plates for gel QC and OD after hyb rxn transfer— the workstation will prepare 2 plates of resuspended samples properly diluted for the fragmentation gel QC and OD quantitation process control checks. See Appendix B, ̋ Fragmentation quality control gel protocolʺ on page 292 and Appendix C, ʺSample quantitation after resuspensionʺ on page 295 for instructions and result assessment guidelines.
• Run method in test mode—select this option to skip all of the incubation timers in a step. If selected, a prompt is displayed asking you to confirm that you want to run a step in test mode (Figure 60). Use this option to perform runs using deionized water only, not actual reagents or samples.
7. Click Start at the top of the left pane to close the default settings window.
IMPORTANT! For troubleshooting and support purposes, we strongly recommend that you perform the gel QC and OD quantitation process controls after resuspension.
CAUTION! Never process samples in test mode. The assay will fail; all of your samples and reagents will be lost.
Figure 60 Prompt displayed when the custom run option, Run method in test mode, is selected.
IMPORTANT! The method preferences (Figure 59 on page 120) should be chosen prior to starting the method. These settings are not prompted for at runtime.
122 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Equipment, consumables, labware, and reagents 4
Equipment, consumables, labware, and reagents
Thermo Fisher Scientific now offers labware consumables kits for target preparation:
• Axiom 96 Consumable Kit for Biomek FXP (Cat. No. 902800)
• Axiom 96 Consumable Kit for QC (Cat. No. 902801)
• Axiom 96 Consumable Kit for Applied Biosystems TC (Cat. No. 902803)
• Axiom 96 TC Plate Sealing Kit (Cat. No. 902802)
Note: Beckman plates, reservoirs, and tips are not included in the kits prepared by Thermo Fisher Scientific. You must order all Beckman supplies directly from Beckman.
Table 30 Axiom 96 Consumable Kit for Biomek FXP (Cat. No. 902800)
Labware item Part No. Quantity
Bio-Rad Hard-Shell 96-well Plate
203015 40
Eppendorf Deep Well Plate 203079 4
Table 31 Axiom 96 Consumable Kit for QC (Cat. No. 902801)
Labware item Part No. Quantity
OD Plate, UV 202609 40
Table 32 Axiom 96 Consumable Kit for off-deck (Applied Biosystems) TC (Cat. No. 902803)
Labware item Part No. Quantity
Bio-Rad Hard-Shell 96-well semi-skirted PCR Plate 203055 5
Costar Clear Polystyrene 96-Well Plates 202165 5
Table 33 Axiom 96 TC Plate Sealing Kit (Cat. No. 902802)
Labware item Part No. Quantity
Bio-Rad Metal Lid 202519 1
Bio-Rad Microseal P Pad 202958 1
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 123
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Equipment, consumables, labware, and reagents4
Labware and materials used on the Biomek workstation deck
Table 34 Guidance for consumable kit ordering
Consumable kit
Thermal cycler option
On-deck,
TRobot
Off-deck PTC-200
Off-deck Applied
Biosystems
Axiom™ 96 Consumable Kit for Biomek FXP (Cat. No. 902800)
Axiom™ 96 Consumable Kit for QC (Cat. No. 902801)
Axiom™ 96 Consumable Kit for Off-Deck (Applied Biosystems) TC (Cat. No. 902803)
Axiom™ 96 TC Plate Sealing Kit (Cat. No. 902802)
Table 35 Labware and materials used on the Biomek workstation deck
Labware Supplier and Cat. No. Image
Biomek AP96 – P250 Pipette Tips (aqua box; pre-sterile, barrier)
Beckman CoulterCat. No. 717253
Biomek Span P250 Pipette Tips (green box; pre-sterile, barrier)
Beckman CoulterCat. No. 379503
124 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Equipment, consumables, labware, and reagents 4
Biomek Span P1000 Pipette Tips (yellow box; pre-sterile, barrier, conductive)
Beckman CoulterCat. No. 987925
Biomek Span P50 Pipette Tips (pink box; pre-sterile, barrier)
Beckman CoulterCat. No. A21586
Bio-Rad Hard-Shell 96-well Plate(available in multiple colors)
Part of the Axiom™ 96 Consumable Kit for Biomek FXP Cat. No. 902800
Plate Part No. 203015
Alternate Vendor:Bio-RadCat. No. HSP-9631
Eppendorf 96 Deep-well Plate, 2,000 μL(available in multiple colors)
Part of the Axiom™ 96 Consumable Kit for Biomek FXP Cat. No. 902800
Plate Part No. 203079
Alternate Vendor:Eppendorf951033481
Table 35 Labware and materials used on the Biomek workstation deck (Continued)
Labware Supplier and Cat. No. Image
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 125
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Equipment, consumables, labware, and reagents4
OD Plate, UV Part of the Axiom™ 96 Consumable Kit for QCCat. No. 902801
Plate Part No. 202609
Alternate Vendor:E & K ScientificEK-25801
Lid, metal(arched, auto-sealing with P pads)
Part of the Axiom™ 96 TC Plate Sealing Kit Cat. No. 902802
Lid Part No. 202519P Pad Part No. 202958
Alternate Vendor:Bio-RadCat. No. MSL-2032 andP Pad Cat. No. MSP-1003
Hard-Shell Full-Height 96-Well Semi-Skirted PCR Plate • This consumable is required only if
using off-deck Applied Biosystems 2720 or Applied Biosystems 9700 Thermal cyclers
Part of the Axiom™ 96 Consumable Kit for Off-deck (Applied Biosystems) TCCat. No. 902803
Plate Part No. 203055
Alternate Vendor:Bio-RadCat. No. HSS-9601
Table 35 Labware and materials used on the Biomek workstation deck (Continued)
Labware Supplier and Cat. No. Image
Front view
Side view
126 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Equipment, consumables, labware, and reagents 4
Plate, Costar Brand Serocluster round Bottom Plate from Corning• Note: this consumable is required
only if using an off-deck Applied Biosystems 9700 or Applied Biosystems 2720 thermal cycle
Part of the Axiom™ 96 Consumable Kit for Off-deck (Applied Biosystems) TCCat. No. 902803
Plate Part No. 202165
Alternate Vendors:VWR International Cat. No. 29442-392E&K Scientific Cat. No. EK 680568Corning Mfg Cat. No. 3795
Beckman Deep Well Titer Plate(polypropylene)
Beckman CoulterCat. No. 267007
Frame for reservoirs Beckman CoulterCat. No. 372795
Table 35 Labware and materials used on the Biomek workstation deck (Continued)
Labware Supplier and Cat. No. Image
Frame
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 127
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Equipment, consumables, labware, and reagents4
Half reservoirHalf module, 75 mL capacity
Beckman CoulterCat. No. 372786
Quarter Reservoirs• Quarter module, 40 mL capacity• Quarter module divided by width,
19 mL capacity each receptacle
Beckman Coulter• Cat. No. 372790 (40 mL)• Cat. No. 372792 (19 mL)
Reagent block, chilled to 4°C Beckman CoulterCat. No. A83054
24-Position Tube Rackwith one 11 mm tube insert in position A6.
Beckman CoulterCat. No. 373661 (rack)Cat. No. 373696 (insert)
Table 35 Labware and materials used on the Biomek workstation deck (Continued)
Labware Supplier and Cat. No. Image
Divided by width19 mL capacity
Undivided40 mL capacity
Metal posts on block circled in red.A1
Tube inserA6
128 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Equipment, consumables, labware, and reagents 4
Adaptor, Deep Well Plate (installed on the Shaking Peltier)
This adaptor is typically installed by a Beckman Coulter field service technician during new system installation or a system upgrade. Ensure that you have one of these adaptors on the deck prior to running this assay.
Beckman CoulterCat. No. A83050
The Applied Biosystems 9700 and the Applied Biosystems 2720 use the semi-skirted 96-well plates (Cat. No. HSS-9601). For use on the Biomek deck, the semi-skirted PCR plate must be stacked on a Costar brand Serocluster 96-well Round Bottom Microtitration plate as shown in the figure to the right.
Reagent Block Template(designed specifically for use with the Axiom 2.0 Reagent Kit)
Contact Thermo Fisher Scientific
Zerostat Anti-static Gunand Ion-Indicator Cap
Milty Zerostat,
Thermo Fisher Scientific Cat. No. 74-0014
Table 35 Labware and materials used on the Biomek workstation deck (Continued)
Labware Supplier and Cat. No. Image
The metal block is the adaptor.
Template on reagent block. Metal posts on block circled in red.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 129
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Equipment, consumables, labware, and reagents4
GeneTitan MC Instrument ConsumablesAll consumables for the GeneTitan MC Instrument are provided by Thermo Fisher Scientific. The following table provides guidance on the consumables that are shipped with the array plate.
IMPORTANT! All covers must have barcodes. Discard any cover without a barcode.
Table 36 Axiom™ GeneTitan™ MC Instrument consumables (Axiom 96-array plate)
Item Part number Labware image Information
Axiom 96-array plate, various designs
Varies, depending on array design.
Axiom 96-Array plate:• Comprised of 3 parts:
clear plastic cover, array plate, and blue array plate protective base.
• The clear plastic cover for the array plate protects the array plate during transport. Discard after opening pouch.
• The array plate must always be kept in the blue array plate protective base at all times.
• The blue array plate protective base in the package holds the array and protects it from damage or exposure to dust.
Note: Array plate is not included in the Axiom GeneTitan Consumables Kit.
Clear tray shipping cover (to be discarded)Array plate protective baseArray plate
1
2
3
1
2
3
130 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Equipment, consumables, labware, and reagents 4
Table 37 Axiom™ GeneTitan™ MC Instrument Consumables (from the Axiom™ Array GeneTitan™ Consumables Kit, Cat. No. 901606)
Item Part number Labware image Information
Scan Tray 900746 Box501006 Pouch
96-Plate Scan Tray:• Comprised of 3
parts: scan tray, black protective base, and a scan tray cover.
• The black scan tray protective base in the package protects the glass bottom of the scan tray from damage before it is loaded into the GeneTitan MC Instrument.
• The scan tray cover protects the contents in the scan tray and must be deionized before used. See Appendix E, "Deionization procedure for GeneTitan™ trays and covers" on page 307.
• Remove the black scan tray protective base before loading the scan tray with the scan tray cover into the GeneTitan MC Instrument.
• The Scan Tray must be loaded into the GeneTitan Instrument with the Scan Tray Cover only.
• Do not load the Scan Tray with the protective base still on.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 131
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Equipment, consumables, labware, and reagents4
Black Scan Tray Protective Base, shown without the Scan Tray with cover
• The black scan tray protective base in the package is used to protect the bottom of the scan tray glass from damage. The black scan tray is distinct from the blue array plate protective base and must not be used with the array plate.
• Remove and set aside the protective base from the scan tray before loading.
Scan Tray with cover, shown without the black protective base
• The GeneTitan scan tray must be loaded with the scan tray cover into the GeneTitan MC Instrument.
• Do not load the scan tray with the protective base.
Table 37 Axiom™ GeneTitan™ MC Instrument Consumables (from the Axiom™ Array GeneTitan™ Consumables Kit, Cat. No. 901606) (Continued)
Item Part number Labware image Information
132 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Equipment, consumables, labware, and reagents 4
GeneTitan 5 Stain Trays Kit
4249910 Kit501025 Tray
• The GeneTitan Stain Tray Kit comes with 5 stain trays packaged in zip-top bags to keep them free of dust.
• The GeneTitan stain trays are barcoded and the trays have separator walls that are flush with the frame of the stain tray, as shown by the yellow line and the yellow oval in the lower photo.
GeneTitan™ Stain and Scan Tray Cover
202757 • The GeneTitan stain and scan tray covers prevent evaporation of the stains in stain trays and the array holding buffer in the scan tray.
• All stain and scan trays must be placed in the GeneTitan MC Instrument with the GeneTitan stain tray cover.
• All tray covers must be deionized to remove static electricity prior to placing the cover on the tray.
• See the section "Deionization procedure for GeneTitan™ trays and covers" on page 307 for the anti-static procedure.
Table 37 Axiom™ GeneTitan™ MC Instrument Consumables (from the Axiom™ Array GeneTitan™ Consumables Kit, Cat. No. 901606) (Continued)
Item Part number Labware image Information
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 133
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Equipment, consumables, labware, and reagents4
Proper tray alignment and loading
Proper alignment and loading of plates, covers and trays is critical when using the GeneTitan MC Instrument. Each plate, cover and tray has 1 notched corner. The notched corner of plates, trays, covers and bases must be in vertical alignment with each other, and placed in position A1 per the Tray Alignment guide inside each GeneTitan MC Instrument drawer (Figure 61 and Figure 62 on page 136).
Note: Tip: Mark the notched corner of each plate, cover and tray with permanent marker to help ensure proper alignment and loading onto the GeneTitan MC Instrument.
Note: The instrument control software will display a warning if it detects a problem during the fluid dispense operations. The filters in the GeneTitan Wash A, Wash B and Rinse bottles should be replaced if the software displays such a warning. See Appendix F, ʺGeneTitan™ Multi-Channel Instrument Careʺ on page 313 for the
GeneTitan stain tray shown with the stain tray cover
Tray 501025Cover 202757
Hybridization Tray
900747 • After aliquoting the denatured hybridization ready samples into the hybridization tray, the tray should be immediately loaded into the GeneTitan MC Instrument with the barcode facing away from the operator, i.e., Barcode should be on the back side.
Table 37 Axiom™ GeneTitan™ MC Instrument Consumables (from the Axiom™ Array GeneTitan™ Consumables Kit, Cat. No. 901606) (Continued)
Item Part number Labware image Information
IMPORTANT! When running a multi-plate workflow, you must pay careful attention to the software prompts that tell you which side of the drawer to place or remove a plate/tray.
CAUTION! Take care not to damage the consumables or bend the blue cover posts or scan tray posts.
134 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Equipment, consumables, labware, and reagents 4
message displayed to the user and the procedure for replacing the filters.
Figure 61 Proper alignment and loading of plates, covers and trays in the GeneTitan MC Instrument
Plates and trays must be seated in this rectangular recess.
The notched corner of all plates, bases, and covers and must be seated in this corner of the drawer per
Tip: Mark the notched corner of each plate, cover and tray with permanent marker to help ensure proper alignment and loading.
Notched corner of array plate aligned with notched corner of blue base.
IMPORTANT! Remove the plastic protective shipping tray cover. 1
2
3
Clear tray shipping cover (to be discarded)Array plate protective baseArray plate
1
2
344
5
5
6
7
7
6
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 135
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Equipment, consumables, labware, and reagents4
Figure 62 Array plate with protective blue base and the hyb tray aligned and properly loaded into drawer 6
Array Plate with Protective Blue Base
Hyb Tray
1
2
1 2
IMPORTANT! When you install the consumables, ensure that the fingers are retracted. Do not lay the consumables on top of the drawer fingers - this indicates that the instrument is not functioning correctly. Notify your field service engineer if the fingers do not retract automatically. You should place the trays into the instrument drawers when a drawer is fully extended by the instrument. The fingers are retracted when the drawer is open and are extended when the drawer is closed in order to restrain the consumable. See Figure 63 for an image showing the location of the tabs.
Figure 63 Photo identifying the location of drawer tabs
2
1
Drawer tab, or “finger” in back.
Drawer tab, or “finger” on right side.
1
2
136 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Equipment, consumables, labware, and reagents 4
Stain trays and covers
Labeling GeneTitan hybridization and reagent traysWhen preparing the hybridization and reagent trays to be loaded onto the GeneTitan MC Instrument, you will need to mark each tray in a way that identifies its contents.
Proper labeling for hyb trays and reagent trays is described in:
• ʺLabeling for hyb traysʺ, below
• ʺLabeling for stain traysʺ on page 139
Labeling for hyb trays
You may label the hyb tray on the front part of the short side of the tray, next to the notch at the left, as shown in Figure 65. The proper section for labeling is closest to the notched corner, corresponding to the A1 and B1 wells.
IMPORTANT! Always place the flat side of the cover against the stain tray.
Figure 64 Placement of covers on trays
Correct placement of cover on stain tray. Incorrect placement of cover on stain tray.
IMPORTANT! It is critical that you write only on the proper locations of the proper sides of hyb and stain trays. Do NOT write in any other location, as this can interfere with sensors inside the GeneTitan MC Instrument and result in experiment failure. To ensure proper placement of lids onto stain trays, and trays onto the GeneTitan MC Instrument, you can also mark the notched corner of the trays and lids.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 137
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Equipment, consumables, labware, and reagents4
Figure 65 Labeling GeneTitan hyb trays
CAUTION! Writing on the wrong side of the hyb tray may interfere with the operation of sensors in the GeneTitan MC Instrument.
Do NOT label trays on the long side of the tray.
Notched corner of the hyb tray should face the front.
Label the hyb tray in this area.
1
2
3
1 2 3
138 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Equipment, consumables, labware, and reagents 4
Labeling for stain trays
You may label the stain trays on the left side of the front of the tray as shown in Figure 66. The correct side is closest to the notched corner, corresponding to the A1 through C1 wells.
IMPORTANT! Do not confuse hyb trays with stain trays.
Figure 66 Labeling GeneTitan stain tray (stain tray shown with lid)
Do NOT label trays on the long side of the tray.
Notched corner of the stain tray should face the front.
Label the stain tray here.
1 2 3
1
2
3
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 139
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Equipment, consumables, labware, and reagents4
Loading tray consumables onto the GeneTitan™ MC Instrument
Loading, or installing, the trays and plates onto the GeneTitan MC Instrument is a simple procedure, but there are certain precautions that you must take in order to ensure an error-free procedure. The following figures show you how to do it. See Figure 67 through Figure 70.
Figure 67 Scan tray loaded in drawer 2
IMPORTANT! When you load the plates, or trays, insert them under the tabs, or fingers, that may protrude into the stage. Confirm that the tray is not resting on these fingers. See Figure 108 on page 211.
Do NOT load the protective black base packaged with the scan tray.
140 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Equipment, consumables, labware, and reagents 4
Figure 68 Stain 1 tray and Ligation tray loaded in drawer 3
Figure 69 Stain 2 tray and Stabilization tray loaded in drawer 4
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 141
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Equipment, consumables, labware, and reagents4
Reagent block template
The Axiom™ 2.0 Reagent Kit template fits precisely onto the top of the chilled reagent block, and is held in place by the metal posts on the block (Figure 71). Using this template will help ensure the proper placement of reagent tubes onto the block for each method.
Reservoir stickers
The reservoir labels are stick-on labels for the modular reagent reservoir frames for the different stages of the automated workflow. These stick-on labels are color-coded to match the colors found on the caps of the reagent tubes in the Axiom 2.0 Reagent Kit. Using these labels helps ensure the proper placement of reservoirs and reagents for each method.
Figure 70 Stain 1 tray loaded in drawer 5
Figure 71 Axiom 2.0 Reagent Template for the reagent block
CAUTION! The reservoir stickers for Windows® 7 (Part No. 101135) are different than the XP stickers. Ensure you are using the correct version.
142 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Equipment, consumables, labware, and reagents 4
There are 4 reservoir holders used in the Axiom 2.0 method. Three of these will have templates on 2 sides and the remaining reservoir holder will have a template on 1 side for a total of 7 templates.
Remove the protective surface from the back of the label and place on the reservoir frames as directed in the Figure 72 through Figure 75.
Reservoir frame 1
Reservoir frame 2
Figure 72 Reservoir frame 1 for Windows® 7 users
Figure 73 Reservoir frame 2 for Windows® 7 users
Side A
Side B
Side A
Side B
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 143
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Equipment, consumables, labware, and reagents4
Reservoir frame 3
Reservoir frame 4
Related Biomek FXP Target Prep Express documentation
The following user manuals are installed at the same time as the Biomek FXP Target Prep Express software (Start All Programs Beckman Coulter Manuals). See these for troubleshooting the Biomek workstation.
• Biomek® Liquid Handler User’s Manual, Beckman Coulter Pub. No. 987834
• Biomek® Software User’s Manual, Beckman Coulter Pub. No. B30026AA
Figure 74 Reservoir frame 3 for Windows® 7 users
Figure 75 Reservoir frame 4 for Windows® 7 users
Side A
Side B
Side A
144 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 1: DNA Amplification 4
Stage 1: DNA Amplification
Note: For this protocol, the term samples includes the positive control.
Duration
Note: A 22–24 hours incubation is required at the end of this stage.
Equipment, consumables, labware and reagents required
Equipment and labware required
IMPORTANT! Before proceeding to DNA Amplification, do the gDNA preparation described in Chapter 2, ʺGenomic DNA preparation and requirementsʺ on page 13.
Table 38 Time required for "Stage 1: DNA Amplification"
Activity Time
Hands-on time ~30 minutes
Biomek FXP Target Prep Express ~19 minutes
Incubation 23 hours
Total time ~24 hours
Table 39 Equipment and labware required
Quantity Item
As required Adhesive seals for plates
1 Benchtop cooler, chilled to –20°C
As required Kimwipes laboratory tissues
1 Marker, fine point, permanent
1 Mini microcentrifuge (microfuge with microtube rotor)
1 Oven (must maintain a constant temperature of 37°C for at least 23 hours with a temperature accuracy of 1°C)• >3 array plates per week—we recommend using the BINDER ED 56• 3 array plates per week—OK to use the GeneChip Hybridization Oven
or the BINDER ED 56
1 Plate centrifuge
1 Vortex
Biomek workstation labware
1 box of each Barrier pipette tips:• Biomek Span P1000 (yellow)• Biomek AP96, P250 (aqua)
2 Plate, Bio-Rad Hard-Shell PCR 96-well
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 145
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 1: DNA Amplification4
Reagents required
2 Plate, deep well titer
1 Reagent block, chilled to 4°C
3 Reservoir, quarter module (40 mL)
2 Reservoir, half module (75 mL)
Table 40 Reagents required for DNA Amplification
Axiom 2.0 Reagent Kit Module
Axiom 2.0 Denat Soln 10X
Module 1, –20°C
Axiom 2.0 Neutral Soln
Axiom 2.0 Amp Soln
Axiom 2.0 Amp Enzyme
Axiom Water
IMPORTANT! The Axiom 2.0 Assay is compatible with only reagents from an Axiom reagent kit. These reagents are not interchangeable with reagents from other Applied Biosystems reagent kits, such as CytoScan, SNP 6.0, DMET Plus, etc.
Table 39 Equipment and labware required (Continued)
Quantity Item
146 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 1: DNA Amplification 4
1. Perform the pre-run checklist
Check the water and waste containers
To check the system water and waste containers:
1. If necessary, fill the system water container using deionized water (no need for ultra-pure water).
2. If necessary, empty the system waste bottle.
Turn on the Biomek FXP Target Prep Express
To turn on the workstation:
1. Power on the workstation.
2. Ensure that all of the peripherals are powered on.
• Two Inheco Single Tec controllers
Each 1 controls the Static Peltier (Pelt_1) or the Shaking Peltier (SPelt_96); no additional power supply.
• Thermal cycler:
Biometra TRobot 96 if present on the Biomek workstation deck. Otherwise, a stand-alone thermal cycler can be used.
3. Launch the Biomek Software by double-clicking the Biomek Software icon on the desktop .
You can also open Start All Programs Beckman Coulter Biomek Software.
Home All AxesThis procedure will home all axes and prime the fluidics lines.
To Home All Axes:
1. Open Instrument Home All Axes.
2. Ensure all conditions in the Warning prompt Figure 76-A are met, then click OK.
An icon instructing you to Stop and wait while the instrument homes is displayed (Figure 76-B).
Figure 76 Homing All Axes
A B
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 147
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 1: DNA Amplification4
3. When:
a. The Warning prompt in Figure 77-A is displayed, confirm that no tips are loaded in the Span-8 Pod, and click OK.
The lines for the Span-8 tips are primed and the next prompt shown in Figure 77-B is displayed.
b. When the intake (syringes and tubing) for the Span-8 tips is clear of bubbles, click OK.
2. Thaw and prepare the reagents and Sample Plate
Thaw and prepare the reagents and Sample Plate
To thaw and prepare the reagents:
1. Thaw the Sample Plate (containing 100 ng or 5 ng/µL of gDNA for human, 150 ng or 7.5 ng/µL of gDNA for diploid plants and animals, or 200 ng or 10 ng/µL of gDNA for polypoid plants and animals) on the benchtop at room temperature and centrifuge.
Note: Do not place a frozen Sample Plate directly on the workstation deck.
2. Thaw the following reagents on the benchtop at room temperature.
• Axiom 2.0 Denat Soln 10X
• Axiom 2.0 Neutral Soln
• Axiom 2.0 Amp Soln
• Axiom Water
Leave the Axiom 2.0 Amp Enzyme in the freezer until ready to use.
Note: Allow ~1 hour for Axiom 2.0 Amp Soln to thaw on the benchtop at room temperature. If the solution is not completely thawed after 1 hour, vortex briefly and return to the benchtop to complete thawing. The bottles can also be thawed in a dish with Millipore water. The Axiom 2.0 Amp Soln must be thoroughly mixed before use.
3. Vortex all reagents (except Axiom 2.0 Amp Enzyme), then place at room temperature.
• Vortex the Axiom 2.0 Amp Soln and Axiom 2.0 Neutral Soln for 30 seconds to thoroughly mix.
• Vortex and centrifuge the Axiom 2.0 Denat Soln 10X before placing on the deck.
• For the Axiom 2.0 Amp Enzyme, just before placing on the deck gently flick the tube 3 times to mix and centrifuge.
4. Preheat the Oven to 37°C.
We recommend using one of these ovens:
• BINDER ED 56
• GeneChip Hybridization Oven 645 (turn rotation on to 15 rpm)
Figure 77 Prompts displayed for priming the Span-8 pod fluidic lines
A B
148 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 1: DNA Amplification 4
3. Run the DNA Amplification step
To run the DNA Amplification step:
1. Open Project Open Project Axiom 2.0 Target Prep and click OK.
2. Open File Open to display the Open Method window.
Or click the Open Method icon .
3. Select Axiom 2.0 Target Prep and click OK
4. At the top of the main window (Figure 78-A), click the Run button to open the Axiom™ Target Prep window.
5. In the Axiom Target Preparation window (Figure 78-B):
a. Select DNA Amplification.
b. Click OK.
The Deck Layout for DNA Amplification is displayed (Figure 79 on page 151).
The Axiom 2.0 Target Prep project folder must be displayed in this menu.
1
1
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 149
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 1: DNA Amplification4
6. Place the labware and reagents on the deck as directed in the following figures and table:
• Figure 79 on page 151—deck layout
• Figure 80 on page 152—location names of empty deck positions
• Table 41 on page 152—table detailing the labware, reagent and modular reservoir placement for the deck layout
• Figure 81 on page 153—reagent block
Figure 78 Opening the Axiom Target Preparation methods window
A B
IMPORTANT! Axiom 2.0 Amp Enzyme—Immediately prior to placing on the reagent block, gently flick the tube with your finger 2 to 3 times to mix; then centrifuge. Do NOT vortex.
150 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 1: DNA Amplification 4
Figure 79 Deck layout window for the DNA Amplification method
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 151
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 1: DNA Amplification4
Figure 80 Empty deck positions
Table 41 Labware and Reagent Locations on the Deck for the DNA Amplification Method
Position on Deck
Labware Reagent or Samples
TL1 Biomek AP96 – P250 Pipette Tips (aqua) —
P1 Beckman Deep Well Titer Plate gDNA samples
P4 Bio-Rad Hard-Shell 96-well plate (any color) —
P10 Reservoirs in frame:• Quarter module (1)• Quarter module (2)• Quarter module (3)
Pour Axiom 2.0 Neutral Solution into Reservoir 2
P11 Reservoirs in frame:• Half module (1)• Half module (2)
• Pour Axiom Water into reservoir 1• Pour Axiom 2.0 Amp Soln into reservoir 2
Pelt_1 Reagent block, chilled to 4°C See Figure 81.
P14 Biomek Span P1000 Pipette Tips (yellow) —
P15 Bio-Rad Hard-Shell 96-well plate (any color)
SPelt96_1 Beckman Deep Well Titer Plate —
1Empty
Reservoir
2Axiom
2.0 Neutral
Soln
3Empty
Reservoir
1
Axiom Water
2
Axiom 2.0 Amp Soln
152 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 1: DNA Amplification 4
7. Check the deck layout to ensure that all labware and reagents are in the proper locations.
Note: If the physical deck does not match the Deck Layout and Confirmation window exactly (Figure 79 on page 151), either modify the physical deck to match exactly or choose Abort in the Deck Layout and Confirmation window.
8. Click OK.
The system flushes the Span-8 fluidics system. Observe the lines and syringes for air bubbles.
Figure 81 Placement of reagents on chilled reagent block for the DNA Amplification step.
Important:
• Position all plates and the chilled reagent block with A1 in the upper left corner of the frame.• centrifuge all reagent tubes prior to placing in block to avoid loss of solution volume to the cap and sides of the tube.• Press reagent tubes into the block to ensure that they are fully seated.
Axiom 2.0Amp
Enzyme
Axiom 2.0 Denat Soln
10X
Reagent block with template.
Only the positions used for this method are shown.
Flick and centrifuge the Amp Enzyme before placing in block.
Diagram of reagent block without template
A1
A1
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 153
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 1: DNA Amplification4
9. At the prompt to repeat the Span-8 fluidics system flush (Figure 82):
• Click No if no air bubbles are present.
• Click Yes if air bubbles are present. Repeat the flush until no air bubbles are present.
The DNA Amplification step runs until the Amplification Master Mix has been added to the Sample Plate. Once complete, the instructions and prompt shown in Figure 83 are displayed.
• Follow the instructions for the Sample Plate (see also ʺUser interventionʺ below).
• Follow the instructions for clearing the workstation deck.
User intervention
1. Remove the Sample Plate.
2. Blot the top of the plate with a Kimwipes laboratory tissue to remove any droplets that may be present.
3. Tightly seal the plate.
4. Vortex and centrifuge the plate.
5. Place in a preheated oven and incubate at 37°C for 23 1 hour.
Note: If using a GeneChip Hybridization Oven, place the plate on the bottom of the oven. Plates do not rotate.
6. After 22–24 hours of incubation, do one of the following:
• Proceed directly to ʺStage 2: Fragmentation and Purificationʺ on page 157
• Tightly seal and store the amplified samples at –20°C.
Figure 82 Flushing the Span-8 fluidics system to purge air bubbles
Figure 83 Instructions for clearing the workstation deck
IMPORTANT! • Seal the Sample Plate before placing in the oven.
• Always discard the used multichannel pipette tips in position P3.
• Always store the reagent block at 4°C.
154 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 1: DNA Amplification 4
Summary of DNA Amplification
Incubate on Deck10 mins @ RT
Stage 1: DNA Amplification
Stage 1: DNA Amplification Page 1
Transfer Denat Soln 1X to Sample Plate
Prepare Denat Soln 1X & Distribution Plate
Span-8
Denat Soln 10X Axiom Water
488 μL 4392 μL
(4°C) (RT)D1 Reservoir
D1 Plate
30 μL/well
D1 Plate Sample Plate
20 μL/well
MC
Continued on AmplificationPage 2
Fill Neutral Soln Plate
N1 Reservoir N1 Plate
140 μL/well
[96] FORMAT TRANSFER MIX MOVE
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 155
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 1: DNA Amplification4
Span-8MC
Continued from AmplificationPage 1
Prepare Amplification Master Mix & Distribution Plate
Amp Soln Amp Enzyme
26374 μL 586 μL
(RT) (4°C)MM Reservoir
Amp Mastermix Plate
255 μL/well
Stage 1: DNA Amplification Page 2
Stage 1: DNA Amplification
Transfer Neutral Soln to Sample Plate
N1 Plate Sample Plate
130 μL/well
Incubate Sample PlateOffline for 23 (±1) hr. @ 37°C
Transfer Amplification Master Mix to Sample Plate
Amp Mastermix
PlateSample Plate
230 μL/well
[96] FORMAT TRANSFER MIX MOVE
Final Volume = 400 mL
156 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 2: Fragmentation and Purification 4
Stage 2: Fragmentation and Purification
Note: Purification is done by precipitation (See Step ʺ4. Precipitationʺ on page 165).
Duration
Equipment, consumables, labware and reagents required
Equipment and labware required
Table 42 Time required for "Stage 2: Fragmentation and Purification"
Activity Time
Hands-on time ~25 minutes~50 minutes if frozen DNA from Step 1
Biomek FXP Target Prep Express– Deactivation incubation—20 minutes to
deactivate the amplification reaction and 20 minutes to equilibrate to the fragmentation temperature
– Fragmentation incubation—30 minutes
1 hour 20 minutes
Total time 1 hour 45 minutes to 2 hours 20 minutes
Table 43 Equipment, consumables and labware required
Quantity Item
As required Adhesive seals for plates
1 Benchtop cooler, chilled to –20°C
1 Freezer, –20°C
1 Ice bucket, filled with ice
As required Kimwipes laboratory tissues
1 Marker, fine point, permanent
1 Mini microcentrifuge (microfuge with microtube rotor)
1 Oven, preheated to 37°C
1 Plate centrifuge
1 Vortex (for plates and microtubes)
Biomek workstation labware
1 box of each Barrier pipette tips:• Biomek AP96, P250 (aqua)• Biomek Span P250 (green)• Biomek Span P1000 (yellow)
2 Plate, Bio-Rad Hard-Shell PCR 96-well
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 157
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 2: Fragmentation and Purification4
Reagents required
1. Perform the pre-run checklist
The following actions are the same as described under ʺ1. Perform the pre-run checklistʺ on page 147. Refer back to this step for details. Some or all of these steps may not be required depending upon the current state of the Biomek workstation.
To perform the pre-run checklist:
1. Power on the Biomek FXP Target Prep Express and all peripherals.
2. Check the water and waste containers; replenish or empty as required.
3. Launch the Biomek Software.
4. Home all axes (on page 147).
1 Eppendorf 96 Deep-well Plate, 2,000 μL
1 Reagent block, chilled to 4°C
3 Reservoir, quarter module
1 Reservoir, quarter module, divided by half
1 Reservoir, half module
Table 44 Reagents required for the Fragmentation Method
Reagent Module
From the Axiom 2.0 Reagent Kit
Axiom Frag Enzyme (leave at –20°C until ready to use)Module 2-1, –20°C
Axiom 10X Frag Buffer
Axiom Precip Soln 2
Axiom Frag DiluentModule 2-2, 2–8°C
Axiom Frag Rxn Stop
Axiom Precip Soln 1
User-supplied
Isopropanol, 99.5% 96 samples: 65 mL
Table 43 Equipment, consumables and labware required (Continued)
Quantity Item
158 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 2: Fragmentation and Purification 4
2. Thaw and prepare the amplified DNA samples and reagents
Thaw and prepare the Amplified DNA Sample Plate
If the plate of amplified samples is frozen:
1. Place the deep well plate in a small water bath.
For example, pour Millipore water into a small tray. Place the frozen plate on the water in the tray.
2. Leave the plate in the water bath for ~50 minutes until all wells have thawed.
3. Centrifuge at 1,000 rpm for 30 seconds.
4. To avoid cross-contamination of wells during vortexing:
a. Remove the seal and blot the top of the plate with a Kimwipes laboratory tissue.
b. Tightly reseal the plate with a fresh seal.
5. Vortex the plate for 30 seconds to thoroughly mix.
6. Centrifuge at 1,000 rpm for 30 seconds.
Thaw and prepare the reagents
To thaw and prepare the reagents:
1. Thaw the following reagents on the benchtop at room temperature.
• Axiom 10X Frag Buffer
• Axiom Precip Soln 2
2. Vortex all reagents (except Axiom Frag Enzyme), then place on ice.
• Vortex and centrifuge Axiom Precip Soln 2 before placing onto deck.
• For the Axiom Frag Enzyme: Leave at –20°C until ready to use. Just before placing on the deck gently flick the tube 3 times to mix and centrifuge.
3. Run the Fragmentation step
To open the Target Preparation methods window:
1. Open Project Open Project Axiom 2.0 Target Prep and click OK.
2. Click the Open Method icon, select Axiom 2.0 Target Prep, and click OK.
3. Click the Run button.
4. Select Fragmentation, then click OK (Figure 84).
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 159
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 2: Fragmentation and Purification4
The deck layout for Fragmentation is displayed (Figure 85).
5. Place the labware and reagents on the deck as directed in the following figures and table:
• Figure 85 on page 161—deck layout
• Figure 86 on page 162—location names of empty deck positions
• Table 45 on page 162—table detailing the labware, reagent and modular reservoir placement for the deck layout
• Figure 87 on page 163—reagent cold block
Figure 84 Selecting the Fragmentation method
IMPORTANT! Remove the seal from the Sample Plate before placing on the deck.
160 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 2: Fragmentation and Purification 4
Figure 85 Deck layout for the Fragmentation method
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 161
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 2: Fragmentation and Purification4
Figure 86 Empty deck positions
Table 45 Labware and reagent locations on the deck for the Fragmentation step
Position on deck
Labware Reagent or samples
TL1 Biomek AP96 – P250 Pipette Tips (aqua) —
P5 Bio-Rad Hard-Shell 96-well plate —
P6 Bio-Rad Hard-Shell 96-well plate —
P7 Beckman Deep Well Titer Plate Amplified gDNA
P8 Eppendorf 96 Deep-well Plate, 2,000 μL —
P10 Reservoirs in frame:• Quarter module (1)• Quarter module (2)• Quarter module, divided (3 and 4)
• Pour Axiom Frag Rxn Stop into reservoir 1• Pour Axiom 10X Frag Buffer into reservoir 2
P11 Reservoirs in frame:• Half module (1)• Quarter module (2)
• Pour Isopropanol into reservoir 1• Pour Axiom Precip Soln 1 into reservoir 2
Pelt_1 Reagent block, chilled to 4°C See Figure 87 on page 163.
P13 Biomek Span P250 Pipette Tips (green)
P14 Biomek Span P1000 Pipette Tips (yellow) —
3Empty
4Empty
1AxiomFragRxnStop
2Axiom10X Frag
Buffer
1
Isopropanol
2AxiomPrecipSol 1
162 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 2: Fragmentation and Purification 4
6. Check the deck layout to ensure that the labware, reagents and samples are in the proper locations.
Note: If the physical deck does not match the Deck Layout and Confirmation window exactly (Figure 85 on page 161), either modify the physical deck to match exactly or choose Abort in the Deck Layout and Confirmation window.
7. Click OK to continue.
The system will automatically flush the Span-8 fluidics system. Observe the lines and syringes for air bubbles.
8. At the prompt to repeat the Span-8 fluidics system flush:
• Click No if no air bubbles are present.
• Click Yes if air bubbles are present. Repeat the flush until no air bubbles are present.
Figure 87 Placement of reagents on chilled reagent block for the Fragmentation step
IMPORTANT:
• Always position the chilled reagent block with A1 in the upper left corner of the frame.• centrifuge all reagent tubes prior to placing in block to avoid loss of solution volume to the cap and sides of the tube.• Press reagent tubes into the block to ensure that they are fully seated.
Axiom Precip Soln 2
Axiom Frag
Diluent
Axiom Frag
Enzyme
Reagent block with template.
Only the positions used for this method are shown.
Diagram of reagent block without template
Axiom Frag Enzyme:
Flick to mix 3 times and centrifuge immediately prior to placing on the chilled reagent block.
A1
A1
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 163
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 2: Fragmentation and Purification4
The Fragmentation step begins. The Sample Plate is incubated at 65°C to inactivate amplification.
9. If you selected Prompt for manual DNA quantitation in the default software settings, you will then be prompted to remove an aliquot of each sample for a optional quantitation process control (Figure 84). The plate will remain at 36.5°C until the aliquots have been collected. Remove 4 µL aliquots from each well into a 96 well PCR plate and set aside for later quantitation (e.g., using the Quant-iT™ PicoGreen® dsDNA Kit from Life Technologies). Immediately continue sample processing by placing the amplification reactions back onto the shaking peltier and clicking OK at the prompt shown in Figure 88.
If Prompt for manual DNA quantitation was not selected, the box in Figure 88 will not appear.
Note: Remain near the Biomek FXP Target Prep Express if you are going to remove aliquots for quantitation. Avoid leaving the samples at 36.5°C for a long period of time.
10. Once the Fragmentation Complete message box appears (Figure 89), follow the instructions in the message box discarding used labware and tips, storing unused tips and storing the cold block at 4°C. Click OK when ready to continue and proceed to Step ʺ4. Precipitationʺ on page 165.
Figure 88 Prompt for manual DNA quantitation
Figure 89 Fragmentation complete message
164 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 2: Fragmentation and Purification 4
4. Precipitation To precipitate the DNA samples:
1. Remove the Precipitation Plate from the deck.
2. Blot the top of the plate with a Kimwipes laboratory tissue and seal tightly.
3. Place the plate in a –20°C freezer overnight to precipitate.
4. Return to the Biomek workstation and clear the deck.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 165
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 2: Fragmentation and Purification4
Summary of Fragmentation
Stage 2: Fragmentation
Span-8MC
Stage 2: Fragmentation Page 1
Inactivate DNA Amplification Reaction
Sample Plate
Incubate:
1) 20 min @ 65.0°C2) 20 min @ 36.5°C
SPelt
Continued on FragmentationPage 2
[96] FORMAT TRANSFER MIX MOVE
Prepare Fragmentation Master Mix & Distribution Plate
Frag Di luent Frag Enzyme
1551.2 μL 150.6 μL
(4°C) (4°C)Frag MM Reservoir
Frag MM Distr ibution Plate
67 μL/well
FragB Reservoir
(RT)
6882.4 μL
166 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 2: Fragmentation and Purification 4
Prepare Precipitation Soln & Distribution Plate
Span-8MC
Continued from FragmentationPage 1
Incubate on Deck
30 mins @ 36.5°C
Transfer Fragmentation Master Mix to Sample Plate
Sample Plate
57 μL/well
Fill Stop Solution Plate
Stop Reservoir Stop Plate
29 μL/well
Precip Soln 2 Precip Soln 1
222 μL
(4°C)
Precip Plate
240 μL/well
Continued on FragmentationPage 3
[96] FORMAT TRANSFER MIX MOVE
Stage 2: Fragmentation
Stage 2: Fragmentation Page 2
FragMM Distribution Plate
Transfer Isopropanol to Precipitation Plate
Iso Reservoir Precip Plate
600 μL/well
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 167
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 2: Fragmentation and Purification4
Stage 2: Fragmentation
Span-8MC
Stage 2: Fragmentation Page 3
Continued from FragmentationPage 2
Transfer Stop Solution to Sample Plate
Stop Plate Sample Plate
19 μL/well
Offline PrecipitationSee user guide
Transfer Sample to Precip Plate
Sample Plate Precip Plate
Plate Contents
[96] FORMAT TRANSFER MIX MOVE
Final Volume = 1316 μL
168 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 3: Centrifugation and drying pellets 4
Stage 3: Centrifugation and drying pellets
Duration
Equipment and consumables required
To centrifuge and dry the pellets:
1. Turn the oven on and preheat to 37°C.
Use an oven that can sustain a constant temperature of 37°C and has a temperature accuracy of 1°C (we recommend the BINDER ED 56). If processing 3 or fewer array plates, you can use a GeneChip Hybridization Oven.
2. Transfer the Precipitation Plate from the –20°C freezer to a pre-chilled centrifuge. Centrifuge the plate for 40 minutes at 4°C at 3,200 x g (4,000 rpm for the Eppendorf 5810R centrifuge with the rotor configuration described in the Axiom™ 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP Site Preparation Guide, Pub. No. 702984).
Table 46 Time required for Centrifugation and drying pellets
Activity Time
Hands-on time 10 minutes
Centrifugation 40 minutes
Drying 25 minutes
Total time 75 minutes
Table 47 Equipment and consumables required
Quantity Item
As required Adhesive seals for plates
As required Kimwipes laboratory tissues
1 Plate centrifuge, precooled to 4°C
1 Oven, preheated to 37°C
Sample Plate One plate of precipitated samples from Stage 2 in an Eppendorf 96 Deep-well Plate
CAUTION! During this step, handle the plate gently to avoid disturbing the pellets. Do not bump or bang the plate.
WARNING! Use rotor buckets with a soft rubber bottom to ensure that the deep well plates do not crack. Do not use buckets where the plates sit directly on a metal or hard plastic bottom, such as the A-4-62 rotor with a WO-15 plate carrier (hard bottom) for the Eppendorf 5810R centrifuge. Use of hard bottom plate carriers may result in cracked plates, loss of sample, unbalanced centrifugation, damage to the instrument and possible physical injury.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 169
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 3: Centrifugation and drying pellets4
3. Immediately after the 40 minutes centrifugation period, empty the liquid from the plate as follows:
a. Remove the seal.
b. Invert the plate over a waste container and allow the liquid to drain.
c. While still inverted, gently press the plate on a pile of Kimwipes laboratory tissues on a bench and leave it for 5 minutes.
4. Turn the plate right side up and place in an oven for 20 minutes at 37°C to dry.
Note: If using a GeneChip Hybridization Oven, place the plate on the bottom of the oven. Plates do not rotate.
5. Do one of the following:
• Proceed directly to ʺStage 4: Resuspension, Hybridization Preparation, and QCʺ on page 171, even if some droplets of liquid remain. Leave the Sample Plate at room temperature. It is helpful to begin preparing reagents for Stage 4 while centrifuging and drying pellets.
• Store the plates for resuspension later in the same day:
• Tightly seal the plates.
• If resuspension will be carried within 4 hours, keep the plates at room temperature.
• If resuspension will be carried out in more than 4 hours, store the plates in a refrigerator (2–8°C).
• Store the plates for resuspension on another day:
• Tightly seal the plate and store at –20°C.
170 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 4: Resuspension, Hybridization Preparation, and QC 4
Stage 4: Resuspension, Hybridization Preparation, and QC
Note: In this stage, resuspension buffer is delivered to the plate of pelleted DNA by the Biomek FXP Target Prep Express. The samples are then resuspended by shaking off deck. The user must pay careful attention to pop-up prompts that inform the user when to remove the plate from the deck to perform an off-deck pellet resuspension step, and when the user must replace the plate with resuspended DNA back on the deck to complete the method.
Duration Resuspension and hybridization preparation
Equipment, consumables, labware and reagents required
Equipment and labware required
Table 48 Time required for Resuspension
Activity Time
Hands-on time 15 minutes
Frozen pellet equilibration to room temperature 1.5 hours
Biomek FXP Target Prep Express 31 minutes
Table 49 Equipment, consumables and labware required
Quantity Item
As required Adhesive seals for plates
1 Benchtop cooler, chilled to –20°C
1 Ice bucket, filled with ice
1 Marker, fine point, permanent
1 Mini microcentrifuge (microfuge with microtube rotor)
1 Plate centrifuge, at Room Temp
1 Vortex
Biomek workstation labware
1 box of each Barrier pipette tips:• Biomek Span P50 (pink)• Biomek AP96, P250 (aqua)• Biomek Span P250 (green)• Biomek Span P1000 (yellow)
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 171
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 4: Resuspension, Hybridization Preparation, and QC4
Reagents required
5 platesOR:
Bio-Rad Hard-Shell PCR 96-well (HSP-9631)For on-deck thermal cycling or when using the PTC-0240/PTC-200 off-deck thermal cycler
6 plates for off deck
cycling with Applied
Biosystems thermal cyclers
4 Bio-Rad (HSP-9631) Hard-Shell PCR 96-well Plate and 1 HSS-9601 Hard-Shell Full-Height 96-Well Semi- Skirted PCR Plate1 Plate, Costar Brand Serocluster round bottom plate
For off-deck thermal cycling using the Applied Biosystems 9700 or Applied Biosystems 2720 thermal cycler– Note: The HSS-9601 plate stacked on the Costar brand serocluster
round-bottom plate should only be used on position P2 on the Biomek FXP deck. See Figure 97 on page 187.
1 Plate, OD
1 Reagent block, chilled to 4°C
1 Reservoir, 19 mL (quarter module, divided)
3 Reservoir, 40 mL (quarter module)
Table 50 Reagents required for Resuspension and Hybridization
Reagent Module
From the Axiom 2.0 Reagent Kit
Axiom Hyb Buffer Module 2-1, –20°C
Axiom Hyb Soln 1
Axiom Resusp Buffer Module 2-2, 2–8°C
Axiom Hyb Soln 2
Other reagents required
Nuclease-Free Water, ultrapure MB grade(Thermo Fisher Scientific, Cat. No. 71786; for OD and gel plate preparation)
To fill line of divided reservoir
TrackIt Gel Loading Buffer, diluted 1,000-fold(see Appendix B, "Fragmentation quality control gel protocol" on page 292 for dilution instructions.)
13 mL
Table 49 Equipment, consumables and labware required (Continued)
Quantity Item
172 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 4: Resuspension, Hybridization Preparation, and QC 4
1. Preparing frozen pellets and Axiom Resusp Buffer
The equilibration of resuspension buffer and pellets to room temperature (18°C to 25°C) is very critical for the success of the Axiom 2.0 assay. When either is cooler than room temperature, pellets may not resuspend completely. This may result in compromised assay performance. Note following guidelines on how to work with plates with fresh, cold or frozen pellets:
Pellets:
• Plates with fresh pellets can be kept at room temperature for up to 1 hour if proceeding with the resuspension and hybridization protocol within an hour.
• Plates with fresh pellets that are not processed within an hour can be transferred to a refrigerator (2-8°C) for few hours only if processed within a day. However, it is critical to equilibrate the plate to room temperature for at least 30 minutes before proceeding with the resuspension and hybridization protocol.
• Plates with frozen pellets (e.g., on Day-5 of 8-plate workflow) must be pre-equilibrated at room temperature for at least 1.5 hour before proceeding with the resuspension and hybridization protocol.
Resuspension Buffer:
• Resuspension buffer, when pulled out from Module 2-2 at 2-8°C, needs at least 1 hour to equilibrate to room temperature.
2. Perform the pre-run checklist
The following actions are the same as described under ʺ1. Perform the pre-run checklistʺ on page 147. Refer back to this step for details. Some or all of these steps may not be required depending upon the current state of the Biomek workstation.
To perform the pre-run checklist:
1. Power on the Biomek FXP Target Prep Express and all peripherals.
2. Check the water and waste containers; replenish or empty as required.
3. Launch the Biomek software.
4. Home all axes (on page 147).
3. Thaw and prepare the reagents
Thaw and prepare the reagents
To thaw and prepare the reagents:
1. Thaw Axiom Hyb Soln 1 and warm Axiom Resusp Buffer on the benchtop at room temperature.
2. Vortex the Axiom Resusp Buffer and the Axiom Hyb Buffer.
3. Vortex and centrifuge Axiom Hyb Soln 1 and Axiom Hyb Soln 2.
4. Run the Resuspension, Hybridization Preparation, and QC step
Note: We strongly recommend that you run 2 quality process controls during this step:
• A gel to verify successful fragmentation
• An OD quantitation of each resuspended sample
The Biomek FXP Target Prep Express can be set to prepare fragmentation and OD plates that are ready for processing. These process controls must be selected as a run preference prior to starting a run. See ʺSetting method preferencesʺ on page 119 for instructions.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 173
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 4: Resuspension, Hybridization Preparation, and QC4
To run the Resuspension, Hybridization Preparation, and QC step:
1. Open Project Open Project Axiom 2.0 Target Prep and click OK.
2. Click the Open Method icon, select Axiom 2.0 Target Prep, and click OK.
3. Click the Run button.The Axiom 2.0 Method selection dialog box appears.
4. Select Resuspension, Hybridization Preparation, and QC and click OK.The deck layout for this method is displayed (Figure 90 on page 175).
5. Place the labware and reagents on the deck as directed in the following figures and table:
• Figure 90 on page 175—deck layout
• Figure 91 on page 176—location names of empty deck positions
• Table 51 on page 176—table detailing the labware, reagent and modular reservoir placement for the deck layout
• Figure 92 on page 177—reagent cold block
6. Label the Bio-Rad plates placed on the deck in positions P2, P9 and P11. For example:
• P2—Hyb Ready + <sample description>
• P9—Dil QC
• P11—Gel QC
Note: Verify the appropriate plastic consumables are being used on the deck for the Hyb Reaction Plate when using the Applied Biosystems 9700 or Applied Biosystems 2720 thermal cycle.
IMPORTANT! The pellets and the resuspension buffer must be at room temperature before proceeding with this step.
174 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 4: Resuspension, Hybridization Preparation, and QC 4
Figure 90 Deck layout for the Resuspension, Hybridization Preparation, and QC
If Prepare plates for gel QC and OD after hyb rxn transfer is selected in method preferences, the following labware is required on the deck:
• Multichannel P50 pipette tips
• Dilution QC plate• Gel QC plate• OD QC plate (do NOT touch
bottom of plate)If not selected, these labware are not needed and will not appear in the deck layout.
See "Setting method preferences" on page 119 for more information.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 175
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 4: Resuspension, Hybridization Preparation, and QC4
Figure 91 Empty deck positions
Table 51 Labware and reagent locations on the deck for Resuspension, Hybridization Preparation, and QC
Positionon deck
Labware Reagent or samples
TL1 Biomek AP96 – P250 pipette tips (aqua) —
P11 Biomek Span P50 Pipette Tip (pink) —
P2 Hyb Reaction Plate:• On-deck TRobot 96 or off-deck PTC-200 or PTC 0240 users:
Bio-Rad Hard-Shell 96-well plate (HSP-9631) or
• Off-deck Applied Biosystems 9700 or 2720 users: Bio-Rad Hard-Shell Full-Height 96-Well Semi-Skirted PCR Plate (HSS-9601) stacked on a Costar Round Bottom plate
WARNING: When using the Applied Biosystems 9700 or the Applied Biosystems 2720 off-deck thermal cycler for denaturing the Hyb Ready Plate, the Bio-Rad HSS-9601 Hard-Shell Full-Height 96-Well Semi-Skirted PCR Plate must be stacked on a CoStar Brand Serocluster Round Bottom Plate (Axiom 96 Consumables kit for off-deck Applied Biosystems users (Part No. 902803)) as shown in Table 35 on page 124.The HSS-9601 and HSP-9631 PCR plates are not interchangeable on the Biomek FXP deck.
P3 Eppendorf 96 Deep-well Plate, 2,000 μL Pelleted samples
P7 Bio-Rad Hard-Shell 96-well plate —
P8 Bio-Rad Hard-Shell 96-well plate
P91 Bio-Rad Hard-Shell 96-well plate Dilution QC Plate
P10 Reservoirs in frame:• Quarter module (1)• Quarter module (2)• Quarter module (3)• Quarter module, divided (4 and 5)
• Pour Axiom Resus Buffer into reservoir 1• Pour Axiom Hyb Buffer into reservoir 2• Leave reservoir 3 empty• Pour Gel Diluent (diluted gel loading buffer) into reservoir 4:
13 mL• Pour nuclease-free water into reservoir 5 to fill line
1Axiom
ResuspBuffer
2AxiomHyb
Buffer
3Empty
Reservoir
4Gel
Diluent
5Water
176 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 4: Resuspension, Hybridization Preparation, and QC 4
P111 Bio-Rad Hard-Shell 96-well plate (for Gel QC) Gel QC Plate
P121 OD plate, 96-well UV OD QC Plate
Pelt_1 Reagent block, chilled to 4°C See Figure 92 on page 177.
P13 Biomek Span P250 pipette tips (green) —
P14 Biomek Span P1000 pipette tips (yellow) —
1 If QC is not selected, leave deck positions P1, P9, P11, and P12 empty.
Table 51 Labware and reagent locations on the deck for Resuspension, Hybridization Preparation, and QC
Positionon deck
Labware Reagent or samples
Figure 92 Placement of reagents on chilled reagent block for Resuspension, Hybridization Preparation, and QC
IMPORTANT:
• Position all plates and the chilled reagent block with A1 in the upper left corner of the frame.• centrifuge all reagent tubes prior to placing in block to avoid loss of solution volume to the cap and sides of the tube.• Press reagent tubes into the block to ensure that they are fully seated.
Axiom Hyb
Soln 2
Axiom Hyb
Soln 1
Reagent block with template.
Only the positions used for this method are shown.
Diagram of reagent block without template
A1
A1
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 177
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 4: Resuspension, Hybridization Preparation, and QC4
7. Check the deck layout to ensure that the labware, reagents and samples are in the proper locations.
Note: If the physical deck does not match the Deck Layout window exactly (Figure 90 on page 175), either modify the physical deck to match exactly or choose Abort in the Deck Layout window.
8. Click OK to continue the method.
The system will automatically flush the Span-8 fluidics system. Observe the lines and syringes for air bubbles.
9. At the prompt to repeat the Span-8 fluidics system flush:
• Click No if no air bubbles are present.
• Click Yes if air bubbles are present. Repeat the flush until no air bubbles are present.
10. There are 2 pop-up prompts that inform the user: 1) when to remove the plate from the deck to perform an off-deck pellet resuspension step, and 2) when the user must return the plate with resuspended DNA back on the deck to complete the method.
Note: Seal the resuspension plate tightly before placing it on the off-deck microplate shaker.
a. The first prompt appears following the addition of the Resuspension Buffer to the DNA pellets (see Figure 93).
• Remove the Sample Plate from Position 3. This plate contains the DNA pellets in Axiom Resuspension Buffer.
b. Follow the instructions below to carry out the DNA pellet resuspension by off-deck shaking. Upon completion of the on-deck method that aliquots the Axiom Resuspension Buffer to the Sample Plate containing the pelleted samples, resuspension is carried out by shaking off-deck using the following steps:
• Blot the top of the Sample Plate using a Kimwipes laboratory tissue to remove any droplets that may be present.
• Seal the plate tightly; blue pellets should be visible at the bottom of the wells.
• Place the Sample Plate onto one of the following shakers for 10 minutes:
– Thermo Scientific Compact Digital Microplate Shaker: set at 900 rpm
– Jitterbug: set at speed of 7
– Inspect the plate from the bottom. If the pellets are not dissolved, repeat the shaking step.
– Centrifuge the plate in a room temperature centrifuge.
c. Ensure that the instructions in the pop-up message box were followed.
d. When ready, click OK to proceed with the method.
The method continues by preparing the Hybridization Master Mix and, if selected, the Sample QC Plates.
Figure 93 Instructions to perform the off-deck DNA pellet resuspension.
178 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 4: Resuspension, Hybridization Preparation, and QC 4
e. A second prompt appears which guides the user to return the Sample Plate back on the deck to prepare the final Hyb Reaction Plate.
f. Centrifuge the Precipitation Plate with resupended DNA samples to get all the droplets down, minimizing sample loss.
g. Unseal the Precipitation Plate before placing it back on the deck at Position 3.
h. Ensure that the instructions in the pop-up message box were followed.
i. When ready, click OK to proceed with the method.
The method continues by preparing the Hyb Reaction Plate (the final hybridization ready samples) and, if selected, the Sample QC Plates.
11. Run the fragmentation gel and OD quantitation process controls.
For instructions and guidelines on assessing gel and OD results, see:
• Appendix B, ʺFragmentation quality control gel protocolʺ on page 292
• Appendix C, ʺSample quantitation after resuspensionʺ on page 295
12. Do one of the following:
• If the GeneTitan MC Instrument is free, and if the gel and OD quantitation results are good:
• Discard used labware and reagents
• Discard used tips
• Store unused Span-8 tips
• Click OK in the Resuspension and Hyb Prep complete dialog box and proceed to ʺStage 5: Preparation for the GeneTitan™ MC Instrumentʺ on page 184.
• If the GeneTitan MC Instrument is not free, then follow the instructions shown in Figure 94:
• Tightly seal the Hyb Rxn Plate and store at –20°C.
• Discard used labware and reagents
• Discard used tips
• Store unused Span-8 tips
• Store cold block at 4°C.
• Click OK when complete.
IMPORTANT! Only proceed with the method once the DNA pellets have been fully resuspended into Axiom Resuspension Buffer using an off-deck microplate shaker.
Figure 94 Instructions to follow if the GeneTitan MC Instrument is not free.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 179
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 4: Resuspension, Hybridization Preparation, and QC4
Summary of Resuspension and Hybridization Preparation
Stage 4: Resuspension & Hybridization Prep
Span-8MC
Stage 4: Resuspension & Hybridization Prep Page 1
Prepare Resuspension Buffer Plate
ResB Reservoir ResB Plate
45 μL/well
Transfer Resuspension Buffer to Precipitation Plate
ResB Plate Precip Plate
35 μL/well
Prepare Hybridization Master Mix & Distribution Plate
Hyb Soln 2 Hyb Soln 1
1197 μL 66.5 μL
(4°C) (4°C)MM Reservoir
Hyb MM Plate
90 μL/well
Hyb Buffer
(RT)
9376.5 μL
Continued on ResuspensionPage 2
[96] FORMAT TRANSFER MIX MOVE
Follow the user interface instructions to perform the
Off-line Pellet Resuspension(See user guide for details)
You must click OKK to continue with method.
A second message will appear later when it is time to place the Sample Plate with resuspended DNA back
on deck.
Final Volume = 35 μL
180 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 4: Resuspension, Hybridization Preparation, and QC 4
Stage 4: Resuspension & Hybridization Prep
Span-8MC
Stage 4: Resuspension & Hybridization Prep Page 2
Continued from ResuspensionPage 1
Transfer Hybridization Master Mix to Precipitation Plate
Hyb MM Plate Precip Plate
80 μL/well
See QC flowchart if performing analysis after Resuspension
Transfer Hybridization Mix to Hyb Reaction Plate
Precip Plate Hyb Rxn Plate
115 μL/well
Store HybridizationReaction Plate
[96] FORMAT TRANSFER MIX MOVE
Final Volume = 115 μL
Follow user interface instructionsto return the sample plate back on the deck
after completing the Off-line Pellet Resuspension(See user guide for details)
Click OKK to continue with method
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 181
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 4: Resuspension, Hybridization Preparation, and QC4
Stage 4: Resuspension & Hybridization Prep QC
Span-8MC
Stage 4: Resuspension & Hybridization Prep QC, Page 1
Fill Dilution QC Plate with Water
Water Reservoir Dilution QC Plate
55 μL/well
Fill OD QC Plate with Water
Water Reservoir OD QC Plate
90 μL/well
Fill Gel QC Plate with Dye
Dye Reservoir Gel QC Plate
120 μL/well
Continued on QCPage 2
Transfer Hybridization Mix to Dilution Plate
Hyb Rxn Plate Dilution QC Plate
5 μL/well
[96] FORMAT TRANSFER MIX MOVE
Final Volume = 60 μL
Wait for the message prompt on whento return the sample plate back on the deck
after completing the Off-line Pellet Resuspension(See user guide for details)
Follow the instructions on the pop-up message and Click OKK to continue with method
182 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 4: Resuspension, Hybridization Preparation, and QC 4
Perform Offline Analysis
Stage 4: Resuspension & Hybridization Prep QC
Span-8MC
Stage 4: Resuspension & Hybridization Prep QC, Page 2
Transfer Diluted Sample to OD QC Plate
Dilution QC Plate OD QC Plate
10 μL/well
Continued from QCPage 1
Transfer Diluted Sample to Gel QC Plate
Dilution QC Plate Gel QC Plate
3 μL/well
[96] FORMAT TRANSFER MIX MOVE
Final Volume = 100 μL
Final Volume = 123 μL
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 183
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 5: Preparation for the GeneTitan™ MC Instrument4
Stage 5: Preparation for the GeneTitan™ MC Instrument
About Stage 5 You will proceed to Stage 5 in 1 of 2 ways:
• Directly from Stage 4 without interruption.
• With samples that were stored at –20°C after Stage 4.
This stage, Preparation for GeneTitan, can include any combination of the options shown in Figure 95. The first 2 options complete target preparation on the Biomek FXP Target Prep Express.
The options are:
Option 1
• Denature samples - the Hyb Rxn plate is placed on the thermal cycler and the samples are denatured. At this step, you must also select the “Transfer denatured samples to hyb tray” option. After the denature program has completed, the block will hold temperature until the user has dismissed the prompt, indicating that they are ready to continue the method to transfer the samples to the hyb tray and then carry it to the GeneTitan MC Instrument. Do not leave samples on the thermal cycler for a long period of time.
Option 2
• Transfer denatured samples to hyb tray - the denatured samples are transferred from the Hyb Rxn plate to the hyb tray, and are ready to load onto the GeneTitan MC Instrument.
Note: When using an Applied Biosystems 9700 or the Applied Biosystems 2720 thermal cycler for off-deck denaturation, the Bio-Rad Hard-Shell Full-Height 96-Well Semi-Skirted PCR Plate (Hyb Reaction Plate) must be stacked on a Costar brand Serocluster Round Bottom plate from Corning (Corning Mfg Cat. No. 3795)
Figure 95 Software setup for Preparation for GeneTitan
You can run one step, or a combination of steps.
184 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 5: Preparation for the GeneTitan™ MC Instrument 4
Option 3
• Prepare GeneTitan™ reagent trays - the solutions required for the fluidics stage of array processing on the GeneTitan MC Instrument are prepared and aliquoted to the appropriate trays (3 stain trays, 1 ligation tray, 1 stabilization tray and 1 scan tray with Holding Buffer).
When performing a 1 plate workflow, select 2 options (option 1 and option 2).
• Denature samples and Transfer denatured samples to hyb tray when you are preparing the samples to begin hybridization in the GeneTitan MC Instrument.
- or select one option (option 3)
• Prepare GeneTitan™ reagent trays to prepare reagents for loading onto the GeneTitan MC Instrument the following day when the plate is finished with the hybridization period and about to begin GeneTitan fluidics processing.
Options for performing a multi-plate workflow
When performing a multi-plate workflow (see Chapter 6, ʺProcessing 8 Axiom™ array plates per weekʺ on page 246 for a description of the 8 plate workflow), the need to carry out preparation of reagents for 1 plate while simultaneously hybridizing a second plate will arise. In this case, all 3 options in the Biomek FXP method can be carried out concurrently. Select all 3 options when using an on-deck thermal cycler.
• Denature samples and Transfer denatured samples to hyb tray will prepare samples for the new plate going into the GeneTitan MC Instrument to begin hybridization.
-and-
• Prepare GeneTitan™ reagent trays will prepare reagents for the plate that is already in the GeneTitan MC Instrument hybridization oven and is ready to go into the Wash, Ligation, Stain, and Scan phases of GeneTitan array processing.
Note: In the 8 plate workflow, all 3 options will only be selected for middle days of the workflow. On the first day of the workflow, only the Denature samples and Transfer denatured samples to hyb tray options will be used. On the last day of the workflow, only the Prepare GeneTitan™ reagent trays option will be used.
Note: When using an off-deck thermal cycler, avoid letting the samples sit at room temperature for an extended period of time after denaturation. Do not begin denaturation at the same time as the GeneTitan reagent preparation.
Off-deck thermal cycler option
The steps for performing a simultaneous preparation of GeneTitan reagent trays and hybridization of a second plate required in the course of a multi-plate workflow are modified slightly when the off-deck thermal cycler option is selected. The reagent trays to be loaded into the GeneTitan MC instrument are prepared in an initial run of the method.
Denaturation of the hybridization ready samples in the off-deck thermal cycler begins mid way through the GeneTitan reagent prep on the Biomek deck.
After loading the reagent trays into the GeneTitan MC instrument, you must perform a second run of the Biomek method to transfer the denatured samples to the hyb tray for loading into the GeneTitan MC instrument. You will need a timer for this modified step.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 185
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 5: Preparation for the GeneTitan™ MC Instrument4
The modified steps are:
1. Select Preparation for GeneTitan step with only the Prepare GeneTitan™ reagent trays box checked, click OK.
2. Prepare deck as shown in Figure 96.
3. Click OK.
4. Start timer. Wait 25–30 minutes, then begin denaturation of the hybridization ready samples using the off-deck thermal cycler.
5. Upon completion of the GeneTitan reagent tray preparation, load reagent trays and scan tray into the GeneTitan MC instrument
6. Once the reagents are loaded into the GeneTitan MC instrument and the denaturation method on the thermal cycler is complete, retrieve the denatured hybridization ready samples from the thermal cycler
7. Return to Biomek FXP and begin a new method, select Preparation for GeneTitan step with only the Transfer denatured samples to hyb tray box checked, click OK.
8. Prepare deck as shown in Figure 97 (note that a spacer must be used with HSS-9601 plates for Applied Biosystems thermal cyclers).
Figure 96 Deck layout for “Prepare GeneTitan™ Reagent Trays” only (selected for Off-deck Thermal Cycler option)
186 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 5: Preparation for the GeneTitan™ MC Instrument 4
9. Click OK.
10. After the denatured samples have been transferred to the GeneTitan hyb tray, load hyb tray into the GeneTitan MC Instrument.
Figure 97 Deck layout for “Transfer Denatured Samples to Hyb Tray”: Off-deck Thermal Cycler option and position P2
-
Bio-Rad Hard-Shell Full-Height 96-Well Semi-Skirted
PCR Plate (HSS-9601) stacked on a Costar Round
Bottom plate for off-deck thermal cycling with the
Applied Biosystems 9700 or 2720 thermal cycler
The Hyb Reaction Plate in deck position P2 must be
one of the following:
Bio-Rad Hard-Shell 96-well plate (HSP-9631) for
on-deck or off-deck thermal cycling using the
TRobot 96, PTC-200 or PTC 0240
OR
--
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 187
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 5: Preparation for the GeneTitan™ MC Instrument4
If a plate is already in the hybridization ovenFor the plate that is already in the GeneTitan hybridization oven and that is ready to go into Ligation, Wash-Stain and Scan, select option 3, Prepare GeneTitan™ reagent trays. The solutions required for the fluidics stage of array processing on the GeneTitan MC Instrument are prepared and aliquoted to the appropriate trays (3 stain trays, 1 ligation tray, 1 stabilization tray and 1 scan tray with Holding Buffer).
Duration of GeneTitan™ MC Instrument reagent preparation and sample preparation
Sample denaturation
Denatured sample transfer to hyb tray
IMPORTANT! The reagent trays prepared in the third sub-step, Prepare GeneTitan™ reagent trays:
• Are NOT for use with the hyb tray currently being prepared on the Biomek workstation.
• Are for the continued processing of an Axiom array plate that is:
– already on the GeneTitan MC Instrument.
– has completed the hybridization stage.
– is ready for transfer to the fluidics area.
The reagent trays for the fluidics stage on the GeneTitan MC Instrument CANNOT be prepared in advance. Do not prepare these plates if there is no array plate ready for the fluidics stage. Once prepared, these plates must be loaded onto the instrument as soon as possible and cannot be stored.
Table 52 Time required to denature samples
Activity Time
Hands-on time: ~3 minutes
Biomek FXP Target Prep Express 17 minutes
Total time ~20 minutes
Table 53 Time required to transfer denatured samples to the hyb tray
Activity Time
Hands-on time 2 minutes
Biomek FXP Target Prep Express ~2 minutes
Total time ~4 minutes
188 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 5: Preparation for the GeneTitan™ MC Instrument 4
Preparation of GeneTitan Reagent Trays
Note: When you select Denaturation and Preparation of the GeneTitan Reagent Trays, the on-deck processes run concurrently.
Equipment and consumables required
Sample Denaturation
Transfer to hyb tray
Table 54 Time Required to Prepare Reagent Trays for the GeneTitan MC Instrument
Activity Time
Prepare reagents (thaw and organize reagents)
~30 minutes
Hands-on time ~15 minutes
Biomek FXP Target Prep Express ~30 minutes
Total time ~75 minutes
Table 55 Equipment required for denaturing sample
If denaturing samples: Item Quantity
• On the Biomek workstation Lid, arched metal 1
Table 56 Consumables required for transferring denatured samples to a hyb tray
Item Quantity
Item required from the GeneTitan™ Consumable Kit, Cat. No. 901606:• hyb tray 1
Pipette tips, Biomek AP96 – P250 (aqua) 1 full box
If using off deck Applied Biosystems 9700 or Applied Biosystems 2720 thermal cyclers: Costar brand Serocluster Round Bottom plate from Corning Mfg. Cat. No. 3795) to be used for stacking under the HSS-9601 semi-skirted plate (Hyb Reaction Plate).
1
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 189
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 5: Preparation for the GeneTitan™ MC Instrument4
Prepare GeneTitan™ reagent trays
Note: See Table 36 on page 130 for GeneTitan MC Instrument Consumable catalog numbers.
1. Perform the pre-run checklist
The following actions are the same as described under ʺ1. Perform the pre-run checklistʺ on page 147. Refer back to this step for details. Some or all of these steps may not be required depending upon the current state of the Biomek workstation.
To perform the pre-run checklist:
1. Power on the Biomek FXP Target Prep Express and all peripherals.
2. Check the water and waste containers; replenish or empty as required.
3. Launch the Biomek Software.
4. Home all axes (on page 147).
Table 57 Consumables and other equipment required
Item Quantity
Items required from the GeneTitan™ Consumable Kit, Cat. No. 901606: • Scan tray with cover and protective base• Stain trays• Covers for stain trays
155
Pipette tips, Biomek AP96 – P250 (aqua) 1 full box
Pipette tips, Biomek Span P1000 (yellow) 1 full box
Pipette tips, Biomek Span P250 (green) 1 full box
Reagent block, chilled to 4°C 1
Reservoirs, quarter module divided 3
Reservoirs, quarter module 3
Tube rack, 24 position with insert (room temperature rack) 1
Zerostat Anti-Static Gun 1
190 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 5: Preparation for the GeneTitan™ MC Instrument 4
2. Prepare the reagents for GeneTitan Reagent Tray Preparation
Note: Ligation Buffer and Ligation Solution 2 require approximately 30 to 40 minutes to thaw on the benchtop at room temperature.
Reagents required
Preparing Axiom Wash A and Axiom Stabilize DiluentDuring storage of the Axiom Wash A and Axiom Stabilize Diluent (in Module 4-2 stored at 4°C), precipitation in the form of clear crystals can sometimes occur. Therefore, follow the procedure below to ensure that any precipitate is returned to solution prior to use.
Note: The presence of some precipitate is okay and will not adversely impact assay performance. Follow the instructions below to resuspend any precipitate before use.
Table 58 Reagents required for GeneTitan MC Instrument Reagent Tray Preparation
Module Reagent Thaw on benchtop, then place on ice
Place on ice Place on benchtop at room temperature
Module 3Room
Temperature
Axiom Wash Buffer A
Axiom Wash Buffer B
Axiom Water
Module 4-1–20°C
Axiom Ligate Buffer - for 30 minutes
Axiom Ligate Enzyme Keep at –20°C until ready to use
Axiom Ligate Soln 1
Axiom Probe Mix 1
Axiom Stain Buffer
Axiom Stabilize Soln
Module 4-22 to 8°C
Axiom Ligate Soln 2 - for 30 to 40 minutes
Axiom Probe Mix 21
Axiom Wash A - for 30 minutes
Axiom Stain 1-A1
Axiom Stain 1-B1
Axiom Stain 2-A1
Axiom Stain 2-B1
Axiom Stabilize Diluent
Axiom Water
Axiom Hold Buffer1
1 These solutions are light sensitive. Keep tubes out of direct light for a prolonged period of time.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 191
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 5: Preparation for the GeneTitan™ MC Instrument4
To prepare the Axiom Wash A:
1. Vortex the bottle for 30 seconds.
2. Place on the benchtop at room temperature for 30 minutes.
3. Examine the reagent for precipitate (look into the top of the bottle).
4. If precipitate is still present, vortex again for 30 seconds.
5. Pour Axiom Wash A into the appropriate reagent reservoir and leave on the benchtop until ready to use.
To prepare the Stabilize Diluent:
If crystals are observed in the Axiom Stabilize Diluent:
1. Vortex and centrifuge.
2. Look for precipitate. If any, warm tube to room temperature and vortex again.
Preparing Axiom Ligate BufferWhite precipitate is sometimes observed when the Axiom Ligate Buffer is thawed.
Note: The presence of some precipitate is okay and will not adversely impact assay performance. Follow the instructions below to attempt to resuspend a majority of precipitate before use.
To prepare the Axiom Ligate Buffer:
1. Place on the benchtop at room temperature for 30 minutes. This bottle can also be thawed in a dish with room temperature Millipore water.
2. Examine the buffer for precipitate (look into the top of the bottle).
3. If precipitate is present, vortex the bottle for 30 seconds.
4. Re-examine the buffer for precipitate.
5. If precipitate is still present, warm the bottle with your hands and vortex again for 30 seconds.
6. If precipitate is still present after hand warming proceed with the protocol below.
7. Pour the Axiom Ligate Buffer into the appropriate reagent reservoir and leave on the benchtop until ready to use.
Prepare the remaining reagents
To prepare the remaining reagents for GeneTitan MC Instrument Plate Preparation:
1. Leave the Axiom Ligate Enzyme at –20°C until ready to use.
2. Thaw the following reagents from Module 4-1 at –20°C on the benchtop at room temperature, then vortex, centrifuge, and place on ice:
• Axiom Ligate Soln 1
• Axiom Probe Mix 1
• Axiom Stabilize Soln
• Axiom Stain Buffer
3. Prepare the remaining reagents from Module 4-2 as follows:
a. Gently flick each tube 2 to 3 times to mix, then centrifuge.
b. Place reagents on ice, except for the Axiom Hold Buffer, Axiom Ligate Soln 2 and Axiom Water— leave these reagents on the benchtop at room temperature until ready to use.
192 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 5: Preparation for the GeneTitan™ MC Instrument 4
3. Prepare the Sample Plate (if stored at –20°C) and the array plate
To prepare samples that were stored at –20°C:
1. Warm the Hyb Ready Plate at room temperature for 5 minutes. It is not necessary to equilibrate the plate for longer duration.
2. Ensure that the Hyb Ready Plate is sealed well. If the plate is not sealed well:
a. centrifuge the plate and carefully remove the old seal.
b. If there is condensation on the top of the plate, blot dry gently with a Kimwipes laboratory tissue.
c. Use a fresh seal and tightly reseal the plate.
3. Vortex the Hyb Ready Plate briefly, then centrifuge to 1,000 rpm.
4. Place the Hyb Ready Plate at room temperature.
To prepare the array plate:
1. Warm up the array plate on the benchtop before setting up hybridization on the GeneTitan MC Instrument.
a. Leave the array plate in the pouch at room temperature, for a minimum of 25 minutes, before opening and loading on the GeneTitan MC Instrument to allow the plate to come to room temperature.
b. At the end of the array warm up time, open the pouch and scan the array plate barcode into the Batch Registration file (see ̋ Stage 1: Create and upload Batch Registration fileʺ on page 219).
4. Prepare the GeneTitan™ MC Instrument
Before your samples are being denatured and/or the Biomek FXP Target Prep Express is preparing reagent trays, ensure that the GeneTitan MC Instrument is ready for use.
One or more of the following steps may need to be performed:
1. Launch GCC and select GCC GeneTitan Control.
2. Upload your sample registration file now, if you have not done so (see ʺStage 1: Create and upload Batch Registration fileʺ on page 219).
If you do not upload your samples before scanning the array plate barcode, the software will assign names to your samples.
3. On the GCC GeneTitan Instrument Control, select the System Setup tab (Figure 98).
4. Configure the software as follows:
a. Setup Option: Hyb-Wash-Scan
Other options available are described under ʺSetup options for array plate processingʺ on page 215.
b. Click Next.
c. Plate Information:
• Barcode: Scan or manually enter the Axiom array plate barcode and click Next.
• Protocol Name: Select the protocol name and click Next.
5. Fill the Wash A, Wash B and Rinse bottles.
WARNING! Do not remove the array plate from the protective base or touch the surface of any arrays.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 193
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 5: Preparation for the GeneTitan™ MC Instrument4
6. Empty the Waste bottle.
• After preparing the GeneTitan MC Instrument, perform steps 6a, 6b or 6c as appropriate for your workflow:
– Go to Step 6a on page 201: if you are denaturing the samples and transferring them to the hyb tray.
– Go to Step 6b on page 202: if you are preparing reagents for the Ligation-Wash-Stain.
– Go To Step 6c on page 202: if you are performing a 2-8 plate workflow, and you are denaturing samples for the 2nd plate and are preparing ligation reagents for the 1st plate. The samples for the 2nd Axiom array plate will be denatured and transferred into the Hyb tray. The Hyb tray will be placed in the GeneTitan MC Instrument with the array plate in preparation for hybridization. The Biomek FXP Target Prep Express will also prepare reagents for Ligation-Wash-Stain for the 1st array plate that was placed in the GeneTitan MC Instrument hybridization oven 24 hours ago.
Figure 98 Setup options for processing array plates.
System Setup tab
Select Hyb-Wash-Scan option
194 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 5: Preparation for the GeneTitan™ MC Instrument 4
5. Run the Preparation for GeneTitan™ step
To run the Preparation for GeneTitan step:
1. Open Project Open Project Axiom 2.0 Target Prep and click OK.
2. Click the Open Method icon, select Axiom 2.0 Target Prep, and click OK.
3. Click the Run button.The Axiom 2.0 Target Prep run options window appears (Figure 99).
4. Select Preparation for GeneTitan™ and the sub-steps that you wish to run; then click OK.
Based upon your choices, the appropriate deck layout is displayed (Figure 100).
5. Deionize the GeneTitan stain trays. See Appendix E, section entitled ʺDeionization procedure for GeneTitan™ trays and coversʺ on page 307 for the deionization procedure.
6. Place the labware and reagents on the deck as directed in the following figures and table:
• Figure 100 on page 196—deck layout
• Figure 101 on page 197—location names of empty deck positions
• Table 59 on page 197—table detailing the labware, reagent and modular reservoir placement for the deck layout
• Figure 102 on page 199—reagent cold block
• Figure 103 on page 200—tube block
Note: Prior to placing the arched metal lid on the deck, be sure to clean the attached Microseal® P pad with 70% ethanol and dry it. See the package insert for this product for further information on cleaning and replacement.
Figure 99 Software setup for Preparation for GeneTitan
You can run each step individually, or any combination of steps. If you do not select Prepare GeneTitan™ reagent trays, the deck layout will be different than the one shown in Figure 100.
• Select Denature Samples and Transfer denatured samples to hyb tray– if you are preparing to load the array plate into the GeneTitan
Instrument for Hybridization.• Select Prepare GeneTitan™ Reagent Trays
– if you are preparing to load the GeneTitan Instrument for Wash-Stain and Imaging.
• Select Denature Samples and Transfer denatured samples to hyb tray and Prepare GeneTitan™ Reagent trays
– if you are running an 8 plate workflow then you will need to select all 3 check boxes in some instances in preparation for the plate that will go into the GeneTitan Instrument for Hybridization.
• Select Prepare GeneTitan™ Reagent trays for the array plate that has completed hybridization and is ready to be processed in the GeneTitan Instrument for Ligation-Wash-Stain and Imaging.
IMPORTANT! It is important to deionize the GeneTitan MC Instrument trays to remove any static electricity on the trays. Static attraction by the trays may prevent the tray cover from being lifted up by the instrument.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 195
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 5: Preparation for the GeneTitan™ MC Instrument4
Figure 100 Deck layout for Preparation for GeneTitan—denature, transfer to hyb tray, and GeneTitan plate preparation
The deck layout displayed is based upon the selections you make for this step.
IMPORTANT: Destatic the stain trays prior to placing them on the deck. See the section "Deionization procedure for GeneTitan™ trays and covers" on page 307.
Included in deck layout if the following options are selected:
• Denature samples• Transfer denatured samples to
Hyb Tray
Included in deck layout if Prepare GeneTitan™ reagent trays is selected.
196 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 5: Preparation for the GeneTitan™ MC Instrument 4
Figure 101 Empty deck positions
Table 59 Labware and Reagent Locations on the Deck for GeneTitan Reagent Preparation
Position on deck
Labware Reagent or samples
If Denature samples and Transfer denatured samples to Hyb Tray are selected, (no reagent tray preparation), only the labware listed below is required:
TL1 Biomek AP96 – P250 pipette tips (aqua) —
P1 Lid, arched metalClean before use (70% ethanol).
—
P2 Hyb Reaction Plate1 Hyb-ready samples
P3 Hyb Tray2 —
If Prepare GeneTitan™ reagent trays is selectd the labware listed below is required:
P4 Stain 12, 3 (second of 2 trays with Stain 1) —
P5 Stain 22, 3 —
P6 Lig2, 3 —
P7 Scan Tray on protective base* —
P8 Stbl2, 3 —
P9 Stain 12, 3 (first of 2 trays with Stain 1) —
P10 Tube block with 1 insert, room temperature See Figure 103 on page 200.
P11 Reservoirs in frame:• Quarter module, divided (1 and 2)• Quarter module, divided (3 and 4)• Quarter module, divided (5 and 6)
• Pour Axiom Water into reservoir 1• Pour Axiom Ligation Buffer into reservoir 5
1Axiom Water
3Empty
5Axiom LigateBuffer
2Empty
4Empty
6Empty
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 197
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 5: Preparation for the GeneTitan™ MC Instrument4
Labeling GeneTitan hybridization and reagent traysWhen preparing the hybridization and reagent trays to be loaded onto the GeneTitan MC Instrument, you will need to mark each tray in a way that identifies its contents.
P12 Reservoirs in frame:• Quarter module (1)• Quarter module (2)• Quarter module (3)
• Pour Axiom Hold Buffer into reservoir 1• Pour Axiom Wash A into reservoir 2• Leave reservoir 3 empty
Pelt_1 Reagent block, chilled to 4°C See Figure 102 on page 199
P13 Biomek Span P250 pipette tips (green) —
P14 Biomek Span P1000 pipette tips (yellow) —
1 For on-deck or off-deck denaturation using the TRobot 96, PTC-200, or PTC 0240, use the Bio-Rad Hard-Shell 96-well plate (Cat. No.HSP-9631). For off-deck denaturation using the Applied Biosystems 9700 or 2720 thermal cycler, use the Bio-Rad Hard-Shell Full-Height96-Well Semi-Skirted PCR Plate (Cat. No. HSS-9601) and place the semi-skirted PCR plate on top of a Costar Round Bottom plate (Cat.No. 3795) for “Transfer Denatured Samples to Hyb Tray”.
2 These trays are included in Axiom Array GeneTitan Consumables Kit (Cat. No. 901606).3 Label each of these stain trays as described above as described in "Labeling GeneTitan hybridization and reagent trays". For example,
label the Stain tray to be placed in P6 with the word “Lig”. This tray will contain the Ligation master mix.
Table 59 Labware and Reagent Locations on the Deck for GeneTitan Reagent Preparation (Continued)
Position on deck
Labware Reagent or samples
1Axiom Hold
Buffer
2Axiom
Wash A
3Empty
Reservoir
IMPORTANT! It is critical that you write only on the proper location of the hyb tray (on the edge in front of wells A1 and B1) as illustrated in Figure 65 on page 138 or on the proper location of the stain/reagent trays (on the edge in front of wells A1 to C1) as illustrated in Figure 66 on page 139. Do NOT write on any other side, as this can interfere with sensors inside of the GeneTitan MC Instrument and result in experiment failure. To ensure proper placement of lids onto stain trays, and trays onto the GeneTitan MC Instrument, you can also mark the notched corner of the trays and lids.
IMPORTANT! Do not confuse hyb trays with stain trays.
198 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 5: Preparation for the GeneTitan™ MC Instrument 4
Figure 102 Placement of reagents on chilled reagent block for GeneTitan Reagent Tray Preparation
IMPORTANT:
• Position all plates and the chilled reagent block with A1 in the upper left corner of the frame.• centrifuge all reagent tubes prior to placing in block to avoid loss of solution volume to the cap and sides of the tube.• Press reagent tubes into the block to ensure that they are fully seated.
Axiom Ligate
Enzyme
Axiom Probe Mix 1
Axiom Probe Mix 2
Axiom Ligate Soln 1
Axiom Stain 2-B
Axiom Stain Buffer
Axiom Stabilize
Soln
Axiom Stabilize Diluent
Axiom Stain 2-A
Axiom Stain 1-B
Axiom Stain 1-A
Reagent block with template.
Only the positions used for this method are shown.
Diagram of reagent block without template
A1
A1
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 199
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 5: Preparation for the GeneTitan™ MC Instrument4
7. Check the deck layout to ensure that the labware, reagents and samples are in the proper locations.
Note: If the physical deck does not match the Deck Layout window exactly (Figure 100 on page 196), either modify the physical deck to match exactly or choose Abort in the Deck Layout window.
8. Click OK to continue the step.
The system will automatically flush the Span-8 fluidics system. Observe the lines and syringes for air bubbles.
9. At the prompt to repeat the Span-8 fluidics system flush:
• Click No if no air bubbles are present.
Click Yes if air bubbles are present. Repeat the flush until no air bubbles are present.
If the Denature Samples option is selected, the Biomek FXP Target Prep Express places the samples onto the thermal cycler and runs the denaturation program. If Prepare GeneTitan™ reagent trays is also selected, reagent tray preparation for the GeneTitan MC Instrument starts while the samples are being denatured.
Figure 103 Placement of reagents on room temperature tube block
AxiomLigationSoln 2
A1
200 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 5: Preparation for the GeneTitan™ MC Instrument 4
6a. Complete Stage 4: Preparation for GeneTitan™ - Hybridization Trays
If you selected Denature samples and Transfer denatured samples to Hyb tray in Step 4 (See Step ʺ5. Run the Preparation for GeneTitan™ stepʺ on page 195 and Figure 99 on page 195), use the following instructions:
1. Once the GeneTitan MC Instrument is ready, return to the Biomek FXP Target Prep Express click OK (prompt shown in Figure 104 above).
The denatured samples are then taken off the thermal cycler and are transferred to the hyb tray.
• Ensure that there are no air bubbles present in the hyb tray. Puncture any air bubbles that you see using a pipette tip.
2. Transfer the hyb tray to the GeneTitan MC Instrument and load. See the section ʺArray processing with the GeneTitan™ MC Instrumentʺ on page 209 for the proper way of loading the array plate and the hyb tray.
3. Return to the Biomek FXP Target Prep Express and clear the deck (Figure 105).
• Always discard the used multichannel pipette tips in position P9.
• Always store the reagent block at 4°C.
• Clean the Microseal P Pad by wiping with 70% EtOH and dry.
See the package insert for this product for further information on cleaning and replacement.
Figure 104 Prompt indicating denaturation is complete.
IMPORTANT! Immediately load the array plate and hyb tray into the GeneTitan MC Instrument.
Figure 105 Instructions displayed at the end of Step 4.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 201
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 5: Preparation for the GeneTitan™ MC Instrument4
6b. Complete Stage 4: Preparation for GeneTitan™ - Reagent Trays
If you selected Prepare GeneTitan™ Reagent trays in Step 4 (See Step ʺ5. Run the Preparation for GeneTitan™ stepʺ on page 195 and Figure 99 on page 195), use the following instructions:
1. Once the prompt shown in Figure 105 appears, remove the reagent trays and scan tray with Hold Buffer from the deck and cover with the appropriate lids.
2. Examine each tray to ensure that:
• All of the wells have been filled. If any wells do not contain reagents, then manually add reagents to these wells.
• There are no air bubbles present. Puncture any air bubbles that you see using a pipette tip.
3. Transfer the reagent trays, scan tray to the GeneTitan MC Instrument and load. See the section ʺStage 3: Ligate, Wash, Stain, and Scanʺ on page 235 to continue the process on the GeneTitan MC instrument.
4. Return to the Biomek FXP Target Prep Express and clear the deck (see Figure 98 on page 194).
• Always discard the used multichannel pipette tips in position P9.
• Always store the reagent block at 4°C.
• Clean the Microseal P Pad by wiping with 70% EtOH and dry.
See the package insert for this product for further information on cleaning and replacement.
6c. Complete Stage 4: Preparation for GeneTitan™ - Multiple Plate Workflow
If you selected all 3 options in Step 4 (See Step ʺ5. Run the Preparation for GeneTitan™ stepʺ on page 195 and Figure 99 on page 195) and are preparing hyb tray and reagent trays in a 2+ plate workflow, use the following instructions:
The Hyb tray is prepared for a new Axiom array plate that will be loaded into the GeneTitan MC Instrument. The reagent trays are prepared for the Axiom array plate that is in the hybridization oven in the GeneTitan MC Instrument and is ready to move to the next stage of the process - Ligation, Wash-Stain and Scan.
Note: When using an off-deck thermal cycler, see the instructions for the ʺOff-deck thermal cycler optionʺ on page 185.
1. Once the reagent trays are prepared and sample denaturation is complete, the prompt in Figure 104 on page 201 is displayed (do not click OK yet). Remove the reagent trays and scan tray with Hold Buffer from the deck and cover with the appropriate lids.
2. Examine each tray to ensure that:
• All of the wells have been filled. If any wells do not contain reagents, then manually add reagents to these wells.
• There are no air bubbles present. Puncture any air bubbles that you see using a pipette tip.
IMPORTANT! Immediately load the reagent trays onto the GeneTitan MC Instrument. Do not leave denatured samples or reagent trays at room temperature for any length of time.
202 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 5: Preparation for the GeneTitan™ MC Instrument 4
3. Transfer the reagent trays, scan tray and array plate to the GeneTitan MC Instrument and load.
4. Return to the Biomek FXP Target Prep Express and click OK at the prompt shown in Figure 104 on page 201.
The denatured samples are then taken off the thermal cycler and are transferred to the hyb tray.
5. Transfer the hyb tray to the GeneTitan MC Instrument and load. See the section ʺArray processing with the GeneTitan™ MC Instrumentʺ on page 209 for the proper way of loading.
6. Transfer the reagent trays, scan tray to the GeneTitan MC Instrument and load. See the section ʺArray processing with the GeneTitan™ MC Instrumentʺ on page 209 to continue the process on the GeneTitan MC Instrument.
7. Return to the Biomek FXP Target Prep Express and clear the deck (Figure 105 on page 201).
• Always discard the used multichannel pipette tips in position P9.
• Always store the reagent block at 4°C.
• Clean the Microseal P Pad by wiping with 70% EtOH and dry.
See the package insert for this product for further information on cleaning and replacement.
IMPORTANT! Immediately load the reagent trays and the hyb tray onto the GeneTitan MC Instrument. Then load the array plate and hyb tray. Do not leave denatured samples or reagent trays at room temperature for any length of time.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 203
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 5: Preparation for the GeneTitan™ MC Instrument4
Summary of Preparation for GeneTitan MC™ Instrument
Stage 5: Preparation for the GeneTitan Instrument
Span-8MC
Stage 5: Preparation for the GeneTitan Instrument Page 1
Move Hyb Lid onto Hyb Rxn Plate
Hyb Lid Hyb Rxn Plate
Fill Scan Tray with Hold Buffer
Hold Buffer Reservoir Hold Buffer Plate
150 μL/well
Continued on GeneTitan Preparation - Page 2
[96] FORMAT TRANSFER MIX MOVE
Prepare Stabilize Solution & Plate
Water Stabi lize Soln
10727 μL 144.96 μL
(RT) (4°C)Stabi lize Solution Reservoir
Stabi lize Solution Plate
105 μL/well
Stabi lize Diluent
(4°C)
1208 μL
Final Volume = 105 μL
Final Volume = 150 μL
204 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 5: Preparation for the GeneTitan™ MC Instrument 4
Stage 5: Preparation for the GeneTitan Instrument
Span-8MC
Stage 5: Preparation for the GeneTitan Instrument Page 2
Continued on GeneTitan Preparation - Page 3
Transfer Wash A Buffer to Stain 1 & 2 Reservoirs
Stain 1 ReservoirStain 2
Reservoir
(RT)
Wash A Buffer
22233.6 μL 11596.8 μL
Continued from GeneTitan Preparation - Page 1
[96] FORMAT TRANSFER MIX MOVE
Transfer Stain Buffer to Stain 1 & 2 Reservoirs
Stain 1 Reservoir Stain 2 Reservoir
(4°C)
Stain Buffer
463.2 μL 241.6 μL
Transfer Stains 1-A & 1-B to Stain 1 ReservoirStain 1-A Stain 1-B
231.6 μL 231.6 μL
(4°C) (4°C)Stain 1 Reservoir
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 205
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 5: Preparation for the GeneTitan™ MC Instrument4
Stage 5: Preparation for the GeneTitan Instrument
Span-8MC
Stage 5: Preparation for the GeneTitan Instrument Page 3
Continued onGeneTitan Preparation - Page 4
Continued fromGeneTitan Preparation - Page 2
Transfer Stains 2-A & 2-B to Stain 2 Reservoir
Stain 2-A Stain 2-B
120.8 μL 120.8 μL
(4°C) (4°C)Stain 2 Reservoir
[96] FORMAT TRANSFER MIX MOVE
Prepare Stain 1 Plates
Stain 1 Plate 1 Stain 1 Plate 2
Stain 1 Reservoir
105 μL/well 105 μL/well
Final Volume = 105 μL
206 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 5: Preparation for the GeneTitan™ MC Instrument 4
Stage 5: Preparation for the GeneTitan Instrument
Span-8MC
Prepare Stain 2 PlateStain 2 Reservoir Stain 2 Plate
105 μL/well
Stage 5: Preparation for the GeneTitan Instrument Page 4
Continued onGeneTitan Preparation - Page 5
Continued fromGeneTitan Preparation - Page 3
Prepare Ligate Solution & Distribution Plate
Ligate Buffer Ligate Enzyme
7610.4 μL 181.2 μL
(RT) (4°C)MM Reservoir
Ligate Plate
105 μL/well
Ligate Soln 1
(4°C) 1510 μL
Ligate Soln 2
362.4 μL (RT)
1208 μL 1208 μL(4°C)(4°C)
Probe Mix 1 Probe Mix 2
[96] FORMAT TRANSFER MIX MOVE
Final Volume = 105 μL
Final Volume = 105 μL
Incubate Hybridization Plate in TRobotHyb Rxn Plate + Lid TRobot
Incubate:
1) 10 min. @ 95°C 2) 3 min. @ 48°C
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 207
Chapter 4 Target preparation on the Biomek FXP with Windows® 7Stage 5: Preparation for the GeneTitan™ MC Instrument4
Stage 5: Preparation for the GeneTitan Instrument
Span-8MC
Stage 5: Preparation for the GeneTitan Instrument Page 5
Unload Hybridization Plate from TRobotHyb Rxn Plate + Lid
Position P2
Remove Lid from Hybridization Plate
Position P2 Position P1
Process Hybridization Trayon the GeneTitan Instrument
Transfer Samples to Hybridization Tray
Hyb Rxn Plate Hyb Tray
105 μL/well
Continued fromGeneTitan Preparation - Page 4
[96] FORMAT TRANSFER MIX MOVE
Final Volume = 105 μL
208 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
5 Array processing with theGeneTitan™ MC Instrument
The Axiom™ 2.0 Assay is designed for processing 96 samples at a time on Axiom™ Genome-Wide and Custom myDesign™ array plates. The protocol is performed in 2 sets of steps:
• Target Preparation: Automated target prep, performed with the Biomek FXP Target Prep Express.
• Array Processing: performed on the GeneTitan Multi-Channel (MC) Instrument.
This chapter includes instructions for Part 2: Array Processing.
Before using the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
Stage 1: Create and upload Batch Registration file . . . . . . . . . . . . . . . . . . . . . . . 219
Stage 2: Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220
Status window prompts and actions required . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
Stage 3: Ligate, Wash, Stain, and Scan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
Continuing the workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
Shutting down the GeneTitan™ MC Instrument . . . . . . . . . . . . . . . . . . . . . . . . . 245
Before using the GeneTitan™ MC Instrument
Note: For optimal GeneTitan MC Instrument performance, the maximum relative humidity must be 80% for temperatures up to 75.2°F (24°C) and a minimum humidity of 30 ±7% relative humidity. Operating outside the working environment specifications leads to higher static levels and results in the evaporation of reagents from stain trays.
Proper tray alignment and loading
Proper alignment and loading of plates, covers and trays is critical when using the GeneTitan MC Instrument. Each plate, cover and tray has one notched corner. The notched corner of plates, trays, covers and bases must be in vertical alignment with each other, and placed in position A1 per the Tray Alignment guide inside each GeneTitan MC Instrument drawer (Figure 106 and Figure 107 on page 211).
Note: Tip: Mark the notched corner of each plate, cover and tray with permanent marker to help ensure proper alignment and loading onto the GeneTitan MC Instrument.
IMPORTANT! When running a multi-plate workflow, you must pay careful attention to the software prompts that tell you which side of the drawer to place or remove a plate/tray.
CAUTION! Take care not to damage the consumables or bend the blue cover posts or scan tray posts.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 209
Chapter 5 Array processing with the GeneTitan™ MC InstrumentBefore using the GeneTitan™ MC Instrument5
Note: The instrument control software will display a warning if it detects a problem during the fluid dispense operations. The filters in the GeneTitan Wash A, Wash B and Rinse bottles should be replaced if the software displays such a warning. See Appendix F, ʺGeneTitan™ Multi-Channel Instrument Careʺ on page 313 for the message displayed to the user and the procedure for replacing the filters.
Figure 106 Proper alignment and loading of plates, covers and trays in the GeneTitan MC Instrument
Plates and trays must be seated in this rectangular recess.
The notched corner of all plates, bases, and covers and must be seated in this corner of the drawer per
Tip: Mark the notched corner of each plate, cover and tray with permanent marker to help ensure proper alignment and loading.
Notched corner of array plate aligned with notched corner of blue base.
IMPORTANT! Remove the plastic protective shipping tray cover. 1
2
3
Clear tray shipping cover (to be discarded)Array plate protective baseArray plate
1
2
344
5
5
6
7
7
6
210 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 5 Array processing with the GeneTitan™ MC InstrumentBefore using the GeneTitan™ MC Instrument 5
Figure 107 Array plate with protective blue base and the hybridization tray aligned and properly loaded into drawer 6
Array plate with protective blue base
Hybridization tray
1
2
1 2
IMPORTANT! When you install the consumables, ensure that the fingers are retracted. Do not lay the consumables on top of the drawer fingers - this indicates that the instrument is not functioning correctly. Notify your field service engineer if the fingers do not retract automatically. You should place the trays into the instrument drawers when a drawer is fully extended by the instrument. The fingers are retracted when the drawer is open and are extended when the drawer is closed in order to restrain the consumable. See Figure 108 for an image showing the location of the tabs.
Figure 108 Photo identifying the location of drawer tabs
2
1
Drawer tab, or “finger” in back.
Drawer tab, or “finger” on right side.
1
2
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 211
Chapter 5 Array processing with the GeneTitan™ MC InstrumentBefore using the GeneTitan™ MC Instrument5
Stain trays and covers
Labeling GeneTitan hybridization and reagent traysWhen preparing the hybridization and reagent trays to be loaded onto the GeneTitan MC Instrument, you will need to mark each tray in a way that identifies its contents.
Proper labeling for hybridization trays and reagent trays is described in:
• ʺLabeling for hybridization traysʺ, below
• ʺLabeling for stain traysʺ on page 214
IMPORTANT! Always place the flat side of the cover against the stain tray.
Figure 109 Placement of covers on trays
Correct placement of cover on stain tray. Incorrect placement of cover on stain tray.
IMPORTANT! It is critical that you write only on the proper locations of the proper sides of hyb and stain trays. Do NOT write in any other location, as this can interfere with sensors inside the GeneTitan MC Instrument and result in experiment failure. To ensure proper placement of lids onto stain trays, and trays onto the GeneTitan MC Instrument, you can also mark the notched corner of the trays and lids.
IMPORTANT! Do not confuse hybridization trays with stain trays.
212 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 5 Array processing with the GeneTitan™ MC InstrumentBefore using the GeneTitan™ MC Instrument 5
Labeling for hybridization trays
You may label the hybridization tray on the front part of the short side of the tray, next to the notch at the left, as shown in Figure 110. The proper section for labeling is closest to the notched corner, corresponding to the A1 and B1 wells.
Figure 110 Labeling GeneTitan hybridization trays
CAUTION! Writing on the wrong side of the hybridization tray, or on the wrong part of the long side, may interfere with the operation of sensors in the GeneTitan MC Instrument.
Do NOT label trays on the long side of the tray.
Notched corner of the hybridization tray should face the front.
Label the hybridization tray in this area.
1
2
3
1 2 3
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 213
Chapter 5 Array processing with the GeneTitan™ MC InstrumentBefore using the GeneTitan™ MC Instrument5
Labeling for stain trays
You may label the stain trays on the left side of the front of the tray as shown in Figure 111. The correct side is closest to the notched corner, corresponding to the A1 through C1 wells.
Email and telephone notifications from the GeneTitan™ MC Instrument
We strongly recommend that you configure the Applied Biosystems™ GeneChip™ Command Console (GCC) software to send you GeneTitan MC Instrument notifications. It is critical that you know when the instrument requires your attention—either for sample handling or troubleshooting. Rapid notification can lessen the risk of sample loss.
Notifications can be sent to email addresses and telephones. See the Applied Biosystems™ GeneChip™ Command Console User Guide for instructions.
The types of notifications available will let you know when a process:
• Starts
• Completes
• Aborts
• Encounters an error
Figure 111 Labeling GeneTitan stain tray (stain tray shown with lid)
Do NOT label trays on the long side of the tray.
Notched corner of the stain tray should face the front.
Label the stain tray here.
1 2 3
1
2
3
214 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 5 Array processing with the GeneTitan™ MC InstrumentBefore using the GeneTitan™ MC Instrument 5
GeneTitan™ MC Instrument lamp
The GeneTitan MC Instrument uses a xenon arc lamp system that is warranted to provide 500 hours of illumination for imaging the array at 2 wavelengths. The xenon lamp has a limited lifetime and needs to be replaced at regular intervals.
The GeneTitan Instrument Control software provides a timer that indicates the remaining useful life of the bulb and notifies you when it requires replacement. It is important to adhere to the warnings specified in the GeneTitan MC Instrument user guide.
See the GeneTitan MC Instrument User Guide, Pub. No. 08-0308, or Appendix F, ʺGeneTitan™ Multi-Channel Instrument Careʺ on page 315 of this user guide for details on replacing the lamp.
See the GeneTitan MC Instrument User Guide, Pub. No. 08-0308, for the Lambda LS and Smart controller system. The Lamp and the controller should NEVER be switched ON or OFF manually. The GeneTitan MC Instrument control software manages the lamp activity and will switch the lamp ON and OFF as required. It takes 10 minutes to warm-up the lamp. In idle mode the lamp will remain ON for 2 hours before it is automatically switched OFF and if there are no more plates being transferred from the fluidics to the imaging station. This is by design and is intended behavior. Do not try to save the lamp life by turning OFF the switch on the lamp.
Note: The power switch on the shutter box should be ON at all times. The OPEN/CLOSE switch on the shutter box should be at AUTO position at all times.
Setup options for array plate processing
The processes (setup options) available for processing array plates are shown in Figure 112. A brief description of each option is given below.
Figure 112 Setup Options for Processing Array Plates
System Setup tab
Setup options
1
2
1
2
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 215
Chapter 5 Array processing with the GeneTitan™ MC InstrumentBefore using the GeneTitan™ MC Instrument5
Hyb-Wash-ScanThis setup option enables you to hybridize, wash-ligate-stain-stabilize, and scan an array plate on the GeneTitan MC Instrument.
• Hyb: the array plate is moved to the hybridization oven inside the instrument. Each denatured sample in the hybridization tray is hybridized to an array on the array plate.
– Duration for 96 samples = 23.5 hours
• Wash: samples on arrays are ligated, washed, stained and stabilized.
– Duration for 96 samples = ~5 hours
Note: The instrument control software will display a warning if it detects a problem during the fluid dispense operations. The filters in the GeneTitan Wash A, Wash B and Rinse bottles should be replaced if the software displays such a warning. See Appendix F, ʺGeneTitan™ Multi-Channel Instrument Careʺ on page 313 for the message displayed to the user and the procedure for replacing the filters.
• Scan: The array plate is moved to the imaging device in the GeneTitan MC Instrument and each array is scanned.
– Duration for 96 samples = ~7.5 hours
Hyb-WashIf this setup option is selected, array plate processing will stop after the array has gone through fluidics processing. Use this option if an array plate cannot be scanned on the same GeneTitan MC Instrument as the one used for hybridization and fluidics processing.
If the array plate cannot be scanned immediately after the Hyb-Wash process is complete:
1. Wrap the array plate (in the scan tray with black protective base) in aluminum foil to protect from light.
No lid is required. Do not invert the plate stack. If inverted, the Hold Buffer will spill out of the tray. To prevent liquid spillage, try to keep the plate level when handling the plates. Do not touch the bottom optical surface of the scan tray.
2. Store at 4°C.
3. Scan the array plate within 3 days or less.
When ready to scan the array plate:
1. Keeping the plate protected from light, bring the plate to room temperature for ~20 minutes.
2. Remove the aluminum foil and load onto the GeneTitan MC Instrument.
Wash-ScanUse this option if:
• You wish to bypass the Hybridization step and perform only the Wash/Stain and Scan steps.
IMPORTANT! When running a multi-plate workflow, you must pay careful attention to the software prompts that tell you which side of the drawer to place or remove a plate/tray.
216 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 5 Array processing with the GeneTitan™ MC InstrumentBefore using the GeneTitan™ MC Instrument 5
Note: It usually takes 25-30 minutes to warm up Wash B if this option is selected.
Wash-Scan-ResumeUse this option if:
• It was necessary to hybridize the array plate in an oven separate from the GeneTitan MC Instrument.
• Fluidics processing has been interrupted (e.g., a power failure occurs at your facility).
ScanUse this option:
• To rescan an entire array plate or specific arrays on a plate that failed to scan for reasons such as bubbles or gridding failure.
• If you have hybridized and performed the fluidics processes on a different GeneTitan MC Instrument than the one you will currently use for the scan, or at a different time.
Unload Plates Use this option to unload plates and trays from the instrument when:
• Array plate processing is complete.
• Array plate processing has been aborted.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 217
Chapter 5 Array processing with the GeneTitan™ MC InstrumentBefore using the GeneTitan™ MC Instrument5
Aborting a process If necessary, you can abort the processing of one or more array plates. Instructions and an example are shown below in Figure 113.
If the instrument aborts a process, you can retrieve the array plate and related consumables as described in Figure 113. An instrument-initiated abort may occur:
• Due to improper placement of plates.
• If the uninterrupted power supply (UPS) detects a long power interruption, draining the UPS to 75% power.
Figure 113 Manually aborting an array plate
To abort array plate processing:
1. Click the Stop button.2. Select the array plate that you want to abort.3. Click Abort.4. Click Yes.5. Wait until the status of the array plate in the
WorkFlow window changes from AbortRequest… to Aborted.
6. Once aborted, retrieve the array plate and other related consumables by:• Using Setup Option: Unload Plates• Loading a new array plate.
Exception: If reagents are loading, abort the plate using the Cancel button displayed in the reagent load step.
Note: If the gripper is required to complete the Abort process, the plate will remain in the “AbortRequest” state until the gripper becomes available.
1
2
3
4
5
218 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 5 Array processing with the GeneTitan™ MC InstrumentStage 1: Create and upload Batch Registration file 5
Stage 1: Create and upload Batch Registration file
You must create and upload a Batch Registration file in the Applied Biosystems™ GeneChip™ Command Console software before you begin ̋ Stage 2: Hybridizationʺ on page 220 (example shown in Figure 114). This file contains information critical for:
• Data file generation during scanning
• Tracking the experimental results for each sample loaded onto an array plate
1. If you have not already created a batch registration file, create one now. (See Appendix D, ʺRegistering samples in GeneChip™ Command Console™ʺ on page 304 for detailed instructions.)
2. In GCC, select the array plate format (96 samples) and open a batch registration file template.
3. Scan the array plate barcode into the yellow barcode field.
4. Enter a unique name for each sample and any additional information.
5. Save the file.
6. Upload the file.
IMPORTANT! It is very important to create and upload a batch registration file with your sample information prior to starting ʺStage 2: Hybridizationʺ on page 220.
Figure 114 Example of a Batch Registration file for an array plate
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 219
Chapter 5 Array processing with the GeneTitan™ MC InstrumentStage 2: Hybridization5
Stage 2: Hybridization
Reagents required Reagents required
An Axiom Genome-Wide Human or non-Human array plate or an Axiom myDesign™ Genotyping 96-Array Plate is required for this step. Prior to inserting this plate into the GeneTitan MC Instrument for hybridization, the array plate should be brought to room temperature as described below:
Warm up the array plate on the benchtop before setting up hybridization on the GeneTitan MC Instrument.
1. Remove the array plate box from the 4°C refrigerator where it is stored.
2. Open the box and remove the pouch containing the array plate and protective base.
3. Leave the array plate in the pouch, unopened but placed on the bench for a minimum of 25 minutes before opening and loading on the GeneTitan MC Instrument to allow the plate to come to room temperature.
4. At the end of the array warm up time, open the pouch and scan the array plate barcode into the Batch Registration file (see ʺStage 1: Create and upload Batch Registration fileʺ on page 219).
A hybridization tray containing denatured samples (from Step 2 on page 106 of Chapter 3) is also required for this step. The samples should be denatured and transferred to the hybridization tray only after the GeneTitan MC Instrument is ready for loading the hybridization tray in the ʺLoad Axiom™ array plate and hybridization tray onto the GeneTitan™ MC Instrumentʺ section on page 225.
Table 60 Reagents required from the Axiom 2.0 Reagent Kit
Reagent Module
Axiom Wash Buffer A(both bottles; 1L) Module 3,
Room TemperaturePart No. 901472
Axiom Wash Buffer B
Axiom Water
WARNING! Do not remove the array plate from the protective base or touch the surface of any arrays.
220 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 5 Array processing with the GeneTitan™ MC InstrumentStage 2: Hybridization 5
Setup the instrument
To setup the instrument:
1. Launch GCC Launcher and select GCC GeneTitan Control (Figure 115).
The system initializes. After initialization, the System Status tab is selected and the status of the Hybridization Oven is displayed at the bottom of the Log window. The status should read: <Time of day> System Ready
Note: The instrument control software will display a warning if it detects a problem during the fluid dispense operations. The filters in the GeneTitan Wash A, Wash B and Rinse bottles should be replaced if the software displays such a warning. See Appendix F, ʺGeneTitan™ Multi-Channel Instrument Careʺ on page 313 for the message displayed to the user and the procedure for replacing the filters.
IMPORTANT! Do not close the scanner application by right-clicking on it and choosing the “Close” option. This will cause the scanner application to exit abnormally and cause undue delay in processing the next plate. The correct way to close the application is described in ʺShutting down the GeneTitan™ MC Instrumentʺ on page 245.
Figure 115 Launching GCC and initializing the GeneTitan MC Instrument
System ready
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 221
Chapter 5 Array processing with the GeneTitan™ MC InstrumentStage 2: Hybridization5
2. Select the System Setup tab (Figure 116).
3. Configure the software as follows:
d. Setup Option: Hyb-Wash-Scan
Other options available are described under ʺSetup options for array plate processingʺ on page 215.
e. Click Next.
Figure 116 System Setup tab and the information displayed in this pane
Status: This field displays the actions that must be performed to prepare or unload the GeneTitan MC Instrument for the setup option that has been selected.
After each action you are instructed to click the Next button or to press the blinking blue Confirmation button located on the GeneTitan MC Instrument.
System Setup tab
Setup Option: The various options you can choose for processing Axiom array plates.
Workflow Steps: This field displays an overview of the user actions required to process an array plate based on the setup option selected.
Barcode: The array plate barcode. Can be scanned or entered manually.
Protocol Name: The protocol that GeneTitan MC Instrument will run. The list of protocols displayed is based on the first 6 digits of the array plate barcode. Only the protocols that are valid for the type of array plate loaded are displayed.
NOTE: If there is not enough disk space, a message is displayed.
• Delete or move .dat files to another location to free up enough disk space for the data that will be generated by 8 Axiom array plates.
• One 96 Axiom array plate requires ~80 GB
222 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 5 Array processing with the GeneTitan™ MC InstrumentStage 2: Hybridization 5
f. Plate Information:
• Barcode: Scan or manually enter the Axiom array plate barcode and click Next.
The first 6 characters of the barcode identify the type of plate being loaded, the protocol GeneTitan MC Instrument will use to process the plate, and the imaging device parameters required for this type of plate.
• Protocol Name: Select the protocol name and click Next.
The system reads the first 6 digits of the array plate barcode to determine which protocols can be run for the type of array plate that has been loaded. Only valid protocols are displayed.
4. Complete the remaining workflow steps as follows:
a. Refill bottles with buffer (Figure 118 on page 224).Fill these bottles:
• Wash A: fill with Axiom Wash Buffer A—keep at 2L full
• Wash B: fill with Axiom Wash Buffer B—Use all 600 mL of Wash B from the reagent kit per Axiom plate. Fill to 1L mark when processing 2 plates on the same day.
• Rinse: fill with Axiom Water—keep at 1L full f
Figure 117 Barcode error message
IMPORTANT! • Always ensure that the GeneTitan bottles containing Wash A and Rinse are above
the 50% mark when setting up the system to process an Axiom array plate. All 600 mL of the Wash buffer B from the Axiom 2.0 reagent Kit should be emptied into the GeneTitan Wash B bottle when setting up the system to process a plate. This ensures that the GeneTitan Wash B bottle is filled to more than the requisite 35% of Wash B bottle volume. Also, do not overfill the bottles. Fill Wash Buffer B and Rinse bottles to the 1L mark only. Wash A keep at 2L. We strongly recommend refilling these bottles every time you are prompted to do so.
If the volume in any of these bottles becomes too low during a run, a message is displayed (see Chapter 8, ʺTroubleshootingʺ on page 281). However, even if you fill the bottle at this time, the instrument may not be able to successfully complete the step that was in progress.
• Wash B—if you intend to load 2 array plates on the same day, fill the Wash B bottle to the 1L mark (use both bottles from the Axiom 2.0 Reagent Kit).
If this error message is displayed:
• Ensure that the library files for the type of array plate you are using are correctly installed.
• Try manually entering the array plate barcode.• Library files must be installed prior to launching the GeneTitan MC Instrument. If a
library file must be installed, exit the GeneTitan MC Instrument, install libraries and relaunch the GeneTitan MC Instrument.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 223
Chapter 5 Array processing with the GeneTitan™ MC InstrumentStage 2: Hybridization5
b. Empty the waste bottle.
c. Press the Confirmation button on GeneTitan MC Instrument to continue. A fluidics check is run (~1 minute).
d. Empty trash bin
• Open the trash bin and empty.
If already empty, the trash bin remains locked and the Status pane reads “Trash bin is empty.”
• Press the Confirmation button to continue.
e. Remove consumable trays and plates
• Remove used trays and plates when drawers open.
• If no consumables to remove, the Status window reads “Drawers are empty.”
• Press the Confirmation button to continue.
f. Continue to ʺLoad Axiom™ array plate and hybridization tray onto the GeneTitan™ MC Instrumentʺ on page 225.
Figure 118 Example of the remaining workflow steps
Workflow step
Specific instructions for each workflow step
224 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 5 Array processing with the GeneTitan™ MC InstrumentStage 2: Hybridization 5
Load Axiom™ array plate and hybridization tray onto the GeneTitan™ MC Instrument
The System Layout pane indicates the position of the various trays in each drawer during a GeneTitan MC Instrument run at maximum throughput. This pane does not change as plates are loaded or removed (Figure 119).
To load an Axiom array plate and hybridization tray onto GeneTitan MC Instrument:
1. When drawer 6 opens, load the array plate and hybridization tray as follows:
a. Examine the wells of the hybridization tray for bubbles; puncture any bubbles with a pipette tip.
b. Load the hybridization tray on the right side of the drawer (Figure 121 on page 226).
c. Remove the array plate and protective blue base from its package.
To avoid dust or other damage, leave the array plate packaged until ready to load onto the GeneTitan MC Instrument (Figure 120).
The array plate must be loaded on its protective blue base, as shown in Figure 121 on page 226. The clear plastic cover on top of the array plate SHOULD NOT be loaded in the GeneTitan MC Instrument. See Figure 106 on page 210 for more details on the correct way of loading the array plate.
Figure 119 System layout—location of plates inside the GeneTitan MC Instrument
IMPORTANT! Removing bubbles at this step greatly reduces the chance of bubbles under the arrays when the hybridization tray and the Axiom array plate are clamped. Bubbles under an array can result in black spots on the array image.
Drawers showing contents.
Each line corresponds to a specific drawer number. In this example “Used Hyb Tray” is in the right side of Drawer 1, and “Hyb Tray” is the right side of Drawer 6.Note: Earlier versions of the software may show
as “Fix Tray” rather than “Stabilizing Tray”.
Right side of drawerLeft side of drawer
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 225
Chapter 5 Array processing with the GeneTitan™ MC InstrumentStage 2: Hybridization5
d. Load the array plate with the protective blue base on the left side of the drawer (Figure 121).
Figure 120 Array plate packaging
Figure 121 Array plate with protective blue base and the hybridization tray properly loaded into drawer 6.
CAUTION! The notched corner of each plate, cover and tray must be aligned. When loading onto the GeneTitan MC Instrument, the notched edge plates, covers and trays must be aligned as indicated by the Tray Alignment guide in the drawer (Figure 121 on page 226).
The error message shown in may be displayed. Plate barcodes must face the internal barcode reader (back of the drawer). Improper tray positioning can cause the GeneTitan MC Instrument to crash, and can result in substantial damage to the instrument and loss of samples.
1
2
3
Clear tray shipping cover (to be discarded)Array plate protective baseArray plate
1
2
3
Array plate with protective blue base Hybridization tray
226 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 5 Array processing with the GeneTitan™ MC InstrumentStage 2: Hybridization 5
e. Press the Confirmation button.
When you load the array plate on the left side of the drawer, the internal bar code reader reads the barcode of the array plate and compares it with the barcode and the plate type specified in the Barcode and Plate Type fields on the Setup page. If the information is correct, the application allows you to proceed to the next step. If the instrument is unable to read the barcode, it will push the tray out and will prompt (Figure 122) you to load the correct plate with the proper orientation into the instrument (Figure 121).
– Click OK to retry and check the loading of the array plate; or
– Click Skip if the instrument has problems reading the barcode and after verifying that the trays have been placed in the proper orientation.
Figure 122 Barcode error message
IMPORTANT! Do not install a 3 plate stack of trays (Figure 123). Confirm that you have removed the clear plastic shipping cover as shown in Figure 106 on page 210.
Figure 123 Do Not install a 3 plate stack of trays
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 227
Chapter 5 Array processing with the GeneTitan™ MC InstrumentStage 2: Hybridization5
f. Select the arrays to scan (instructions in Figure 124).
By default, all arrays are selected.
2. Click Next, then click OK to begin processing the samples.
The array plate is placed on top of the hybridization tray and clamped (now referred to as the plate stack).
The software starts the process for clamping the array plate to the hybridization tray. Press OK on the dialog shown in Figure 125 and wait for the drawer to open before retrieving the array plate and hybridization tray combo for inspection. The sandwich of the array plate and hybridization tray needs to be manually inspected before the array processing can begin. Once clamping is complete the dialog shown in Figure 126 on page 229 will be displayed. If you do not press OK in Figure 125 the dialog box will go away without intervention and Figure 126 on page 229 will be displayed.
Figure 124 Selecting which arrays to scan on an array plate
Figure 125 Clamping in progress notification
Default – all arrays are selected
Single array - click one box
Multiple arrays:
– Click one box– Hold down the Ctrl key– Click another box in the same column
Group of arrays:
– Click one box– Hold down the Shift key– Left-click and drag the mouse
Message in Status window.
228 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 5 Array processing with the GeneTitan™ MC InstrumentStage 2: Hybridization 5
3. When drawer 6 opens and the prompt in Figure 126 is displayed:
a. Remove the plate stack and gently press the 2 plates together at each clamping point.
Listen for a clicking sound which indicates that the plates are now clamped. No clicking sound indicates the plates are already clamped. (The figure below shows an example of an array plate and hybridization tray stack.)
Figure 126 Location of camping points on the array plate and hybridization tray
Clamping points on an Axiom array plate and hybridization tray
Array Plate
Hybridization tray
Notched corners
1 1
1 1
1 1
1
2
3
4
2
3
4
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 229
Chapter 5 Array processing with the GeneTitan™ MC InstrumentStage 2: Hybridization5
b. Inspect the bottom of the plate stack for bubbles under the arrays—do NOT invert the plates.
c. If bubbles are present, gently tap the plate until the bubbles move out from under the arrays—do NOT unclamp the plate stack.
d. Return the plate stack to the drawer, and press the Confirmation button to proceed.
The message below may be displayed again if plate orientation is incorrect or if the hybridization tray barcode cannot be read. Click OK to proceed.
230 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 5 Array processing with the GeneTitan™ MC InstrumentStage 2: Hybridization 5
Load a second Axiom™ array plate and hybridization tray onto the GeneTitan™ MC Instrument
When you can load a second array plate and hybridization trayOnce processing begins, you have a specific period of time during which you can load another Axiom array plate and hybridization tray. This period of time is displayed above the Hyb Oven Status pane (Figure 127). You cannot load another hybridization tray before or after this period of time.
While the first plate is in the oven, you can load another plate if the time spacing requirement is met. This is to ensure that the second plate does not have to wait for system resources in its workflow. The time spacing is roughly equal to the longer of the wash-stain or scan time of the first plate.
IMPORTANT! You must load the next array plate and hybridization tray during the period of time displayed above the Hyb Oven Status. You cannot load another hybridization tray before or after this period of time. You will have to wait until the current process is finished which will result in disruption of the 8 plate workflow and fewer than 8 plates processed per week.
Figure 127 Loading a second hybridization tray based on hybridization oven status information
This pane displays the period of time during which another array plate and hybridization tray can be loaded.
Additional plates cannot be loaded before or after this period of time while the instrument is operating.
In this example the system is currently available.
Position of plate stack in the hybridization oven.
Position 1 - left side of oven
Position 2 - right side of oven
Oven Temperature.
• Green indicates the current oven temperature is within the target temperature range.
• Yellow indicates oven temperature outside of target temperature range.
1
2
3
1
2
3
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 231
Chapter 5 Array processing with the GeneTitan™ MC InstrumentStage 2: Hybridization5
1. Select the System Setup tab.
2. Load an Axiom array plate and hybridization tray in the same manner that you loaded the previous plate and tray.
a. Scan or manually enter the Axiom array plate barcode, then click Next.
b. Load the Axiom array plate with the blue base (without the cover) and the hybridization tray (as shown in Figure 121 on page 226), then press the Confirmation button.
c. Select the arrays to scan, then click Next.
d. Ensure that the plates are clamped securely when prompted, then press the Confirmation button.
e. Click OK when prompted to resume plate processing.
Select the System Status tab to view Axiom array plate status in the WorkFlow window (Figure 128).
Figure 128 Example of the workflow window when 2 plates are loaded and are in the hybridization oven
Location: Left and Right positions = the position of the scan tray in drawer 2 (left or right side of the drawer).
1
1
232 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 5 Array processing with the GeneTitan™ MC InstrumentStatus window prompts and actions required 5
Status window prompts and actions required
As a part of normal GeneTitan MC Instrument operations you may see the following status prompts. Table 61 explains the necessary actions required.
Table 61 Refilling buffer bottles and emptying the waste bottle
Status window prompt Action required Receptacle – Reagent
Buffer bottles have been depressurized. Refill buffer into the bottles. Empty the waste bottle.
• Replenish the fluid in Wash Bottles A and B, and the Rinse bottle.1
• Empty the Waste Bottle.• Press the Confirmation button to
continue.
• Wash Bottle A – fill with Axiom Wash Buffer A up to 2L.
• Wash Bottle B – fill with Axiom Wash Buffer B to the 1L mark.
• Rinse – fill with Axiom Water to the 1L mark.
Do not overfill these bottles.
1 Every time you are prompted to refill the buffer bottles, the system runs a fluidics check (duration ~1 minute).
Table 62 Emptying the trash bin
Status window prompt Action required Receptacle – Reagent
Empty trash bin • Open and empty the trash bin.• Press the Confirmation button to
continue.
Note: If the trash bin is empty, you will not be able to open it. Continue the process by pressing the blue confirmation button
—
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 233
Chapter 5 Array processing with the GeneTitan™ MC InstrumentStatus window prompts and actions required5
Table 63 Loading the array plate and hybridization tray; barcode error messages
Status window prompt Action required Reagent – Receptacle
Load array plate tray on [Left/Right] side of drawer. Load hybridization tray without cover on [Left/Right] side of drawer.
Load the array plate with the blue base and the hybridization tray in drawer 6.• IMPORTANT! The blue base must remain in “left side
HTA in” even when empty.• IMPORTANT! The trays must be positioned correctly. If
the trays are placed incorrectly, the software will display an error dialog box indicating the barcode could not be read.
• Press the Confirmation button to continue.
• Hybridization tray loaded with denatured samples.
These messages are displayed if:• A plate has been loaded
improperly.• The bar code is missing
or obscured
Text version of the error message
WARNING! The system was not able to verify the array plate barcode.
Verify that the tray on the left side of the drawer has a blue protective base and if applicable, an array plate, in the correct ORIENTATION. The right side of the drawer should contain a hybridization tray, if applicable, in the correct ORIENTATION.
Details:
The consumable is either not the correct consumable, not loaded correctly, or its barcode is not readable. Proceeding with an incorrect or incorrectly loaded consumable can result in a loss of consumables, loss of samples and may require a Field Service Engineer to service the instrument.
See the System Setup tab or the user guide provided with the Assay or GCC for instructions on proper consumable placement.
Press the flashing blue confirmation button or...
Press OK, the GeneTitan MC Instrument will verify the barcode and orientation.
Press Skip, the GeneTitan MC Instrument will NOT verify the barcode and orientation. The barcode entered at registration will be used.
Table 64 Selecting which arrays to scan
Status window prompt Action required Reagent – Receptacle
Select arrays to scan • Accept the default (all arrays selected) if appropriate. Otherwise, select the arrays to be scanned.
• Click Next, then click OK to start processing.
—
234 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 5 Array processing with the GeneTitan™ MC InstrumentStage 3: Ligate, Wash, Stain, and Scan 5
Stage 3: Ligate, Wash, Stain, and Scan
Equipment, consumables, and reagents required
Scan tray with Axiom Hold Buffer• Cover the tray by orienting the notched corner of the cover over the notched edge
of the tray and leave on the benchtop (no need to protect from light).
CAUTION! Do not remove the scan tray from its protective black base. Leave the scan tray in the base until loaded onto the GeneTitan MC Instrument. When handling the scan tray, the bottom glass surface of the tray should not be touched.
Notched corner of the cover is aligned with the notched corner of the scan tray.
Always leave the scan tray in its protective black base.
1
1
2
2
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 235
Chapter 5 Array processing with the GeneTitan™ MC InstrumentStage 3: Ligate, Wash, Stain, and Scan5
Proper installation of the GeneTitan™ tray consumables
It is very important that you load the GeneTitan tray consumables in the proper orientation. The barcode faces into the instrument (see Figure 129 and Figure 130).
Note: The instrument control software will display a warning if it detects a problem during the fluid dispense operations. The filters in the GeneTitan Wash A, Wash B and Rinse bottles should be replaced if the software displays such a warning. See Appendix F, ʺGeneTitan™ Multi-Channel Instrument Careʺ on page 313 for the message displayed to the user and the procedure for replacing the filters.
Figure 129 You must rotate and load the trays so that the barcode faces into the instrument.
Figure 130 The proper loading of the GeneTitan Tray consumables is shown(the image shows the Stain Tray and the Stain Tray cover as an example).
Notch(This faces out and left)
Barcode(This faces BACK TO THE REAR of the instrument)
Turn the tray and cover combo so that the barcodes face BACK AND INTO the instrument and the notch faces OUT AND TO THE LEFT.
FRONT(OF INSTRUMENTFACING YOU)
Notch faces out and left.“For Research Use Only” faces out.
Barcode faces in and back.
FOR RESEARCHUSE ONLY
FOR RESEARCHUSE ONLY
236 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 5 Array processing with the GeneTitan™ MC InstrumentStage 3: Ligate, Wash, Stain, and Scan 5
Load trays onto the GeneTitan™ MC Instrument
To load trays onto the GeneTitan MC Instrument:
When hybridization of an Axiom array plate has finished, a message alerts you to resume the workflow setup. Press OK and the software takes you directly back to the System Setup tab.
This prompt to continue into reagent load occurs when the hyb is complete. “Estimated Time Remaining” displayed under “Hybridization Oven Status” may display a time remaining of 0 to 30 minutes when the prompt occurs.
The GeneTitan MC Instrument will allow reagent load to take place after either:
• the estimated time counts down to zero, or
• the actual real world hyb time (as indicated by the computer clock) indicates the hyb is complete.
Note: The time estimate displayed on some systems may lag due to high CPU utilization. The GeneTitan MC Instrument allows the workflow to synchronize with the system clock to compensate for this situation during the final half hour of the hyb time estimate. When this prompt to resume reagent loading is displayed to the user there is no need to wait for the estimated time to count down to zero.
Follow the prompts displayed to continue with staining, ligation, stabilizing and scanning.
1. Follow the prompts in the Status window.
a. Wash Bottles A and B, and the Rinse Bottle—refill as necessary (the system will prime itself again); Waste bottle—empty if necessary.
• Wash bottle A—2L
• Wash Bottle B and Rinse Bottle—fill to 1L mark only.
b. Empty the trash bin.
c. Remove consumable trays and plates as instructed, except for the blue base.Leave the blue array plate base in drawer 6 even though the base is empty.
2. Load consumable trays and plates as follows:
a. Follow the prompts in the Status window (load sequence and prompts in Table 65).
b. Once loaded, examine each cover for droplets of liquid.
c. If any liquid is present, remove the tray, clean the cover and top of the tray with Kimwipes laboratory tissues, and reload the tray.
CAUTION! • Orient trays as indicated by the guide inside the drawer. Improper
orientation may cause the run to fail.
• Remove the protective black base from the scan tray immediately prior to loading Figure 131 on page 239).
• Examine each cover for droplets of liquid after loading. Liquid on the cover can result in capillary phenomenon. As a result, the tray may stick to the cover and be lifted out of place inside the instrument.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 237
Chapter 5 Array processing with the GeneTitan™ MC InstrumentStage 3: Ligate, Wash, Stain, and Scan5
Table 65 Sequence for loading the trays with reagents
Loading sequence by
drawer number
Left Right
Note: If the software is unable to verify the barcode on the scan tray and the scan tray cover, the software will display the following error message.
2 Scan Tray with cover—do not load the protective black base(left side of drawer as indicated in Status window)
Figure 131 on page 239
3 Stain Tray with Stain 1 Ligation Tray
Figure 132 on page 240
4 Stain Tray with Stain 2 Stabilization Tray with Stabilization Reagent
Figure 133 on page 241
238 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 5 Array processing with the GeneTitan™ MC InstrumentStage 3: Ligate, Wash, Stain, and Scan 5
5 Stain Tray with Stain 1 Empty
Table 65 Sequence for loading the trays with reagents (Continued)
Loading sequence by
drawer number
Left Right
Figure 131 Scan tray loaded in drawer 2
Do NOT load the protective black base packaged with the scan tray.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 239
Chapter 5 Array processing with the GeneTitan™ MC InstrumentStage 3: Ligate, Wash, Stain, and Scan5
IMPORTANT! When you load the plates, or trays, insert them under the tabs, or fingers, that may protrude into the stage. Confirm that the tray is not resting on these fingers.
1
1
1 Tab or “finger” in GeneTitan drawer.
Figure 132 Stain 1 tray (left) and Ligation tray (right) loaded in drawer 3
240 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 5 Array processing with the GeneTitan™ MC InstrumentStage 3: Ligate, Wash, Stain, and Scan 5
3. At the prompt, click Yes to load another Axiom array plate and hybridization tray. Right or left position is determined by the position of Axiom array plates already in the GeneTitan MC Instrument.
4. Follow the prompts and:
a. Setup Option: select Setup Another Run, then click Next.
b. Scan or manually enter the Axiom array plate barcode, then click Next.
c. Select a protocol, then click Next.
Figure 133 Stain 2 tray (left) and Stabilization tray (right) loaded in drawer 4.
Figure 134 Stain 1 Tray loaded in drawer 5
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 241
Chapter 5 Array processing with the GeneTitan™ MC InstrumentStage 3: Ligate, Wash, Stain, and Scan5
d. When drawer 6 opens:
• Remove the blue base from the previous Axiom array plate.
• Load a new Axiom array plate and new blue base on the left; load a new hybridization tray on the right.
• Press the Confirmation button.
e. Click OK when prompted in the Confirm Resume Processing message.
f. When drawer 6 opens, confirm that the plate stack is securely clamped, then press the Confirmation button.
The following is a description of array plate movements in the GeneTitan MC Instrument as users execute a multi-plate workflow.
1. The plate stack which has finished hybridization is moved from the hybridization oven to drawer 1 (temporarily).
2. The new plate stack in drawer 6 is moved to the hybridization oven.
3. The plate stack currently in drawer 1 (see Step 1) is moved to the unclamping station where it is unclamped and moved into the fluidics section of the GeneTitan MC Instrument.
Note: At the end of a Hyb-Wash-Scan run, all plate and tray covers should be in the trash.
Figure 135 is an example of how the System Status Workflow window appears when 3 Axiom array plates are being processed.
242 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 5 Array processing with the GeneTitan™ MC InstrumentStage 3: Ligate, Wash, Stain, and Scan 5
Figure 135 Example of the System Status window—3 Axiom array plates are being processed
Workflow indicates the number of plates being processed and where they are in the instrument. In this example, 3 Axiom array plates are being processed: 2 in the hybridization oven and 1 in fluidics.Estimated Completion Time is for the current process.
Status area: Current status indicates that another (4th) plate cannot be added to the GeneTitan hybridization oven because both oven slots are currently in use.
Estimated Time Remaining for fluidics is adjusted as necessary. Adjustments can be due to process interruptions such as a drawer being opened.
Step currently executing in fluidics.
1
1
2
2
3
4
3
4
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 243
Chapter 5 Array processing with the GeneTitan™ MC InstrumentContinuing the workflow5
Continuing the workflow
Once a plate has gone through the fluidics stage of the process, it is moved to the imaging device.
When the scanning process begins, the window shown in Figure 136 is displayed. This window must remain open while Axiom array plates are being scanned.
CAUTION! • The Scan Control window must remain open while Axiom array plates are
being scanned. Closing this window will halt the scanning process. You can minimize this window if necessary without creating any interference to the imaging.
• Do not manually, or through the GCC transfer utility, move any data associated with the current plate that is being processed/scanned. Transferring data will dramatically slow scanning and may cause the computer to freeze.
Figure 136 Scan Control window
244 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 5 Array processing with the GeneTitan™ MC InstrumentShutting down the GeneTitan™ MC Instrument 5
Shutting down the GeneTitan™ MC Instrument
This procedure assumes that all of the Axiom array plates loaded onto the GeneTitan MC Instrument have been processed.
To Shutdown the GeneTitan MC Instrument:
1. On the System Setup page, open the Setup Options drop-down menu and select Unload Plates.
2. Unload all of the consumables as prompted.
3. Power off the GeneTitan MC Instrument by opening Tools Shutdown from the menu.
4. Exit the Applied Biosystems™ GeneChip™ Command Console software if it does not close automatically.
Note: If the instrument is processing an array plate, the software will not allow you to shut down the system.
WARNING! Do not attempt to shut down the GeneTitan MC Instrument while array plates are being processed.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 245
6 Processing 8 Axiom™ array platesper week
Using 1 Biomek FXP Target Prep Express (Biomek workstation) and 1 GeneTitan MC Instrument, the Axiom™ 2.0 Genotyping Assay (using the 96-array plate) can be run at a throughput of 8 Axiom™ array plates per 5-day work week. This chapter includes tables that present the timing of the steps required to perform this workflow per 5-day work week, 8 hours per day.
Overview of the 8-plate workflow
During the initial week of startup, all work is done on the Biomek FXP Target Prep Express. You will process 8 plates of genomic DNA samples. At the end of this week, you will have what are now referred to as 8 plates of hyb-ready target (target).
Subsequent weeks of the workflow involve the simultaneous processing of plates on the Biomek FXP Target Prep Express and on the GeneTitan MC Instrument. Each week:
• Eight plates of target from the previous week are denatured and transferred to the GeneTitan MC Instrument for hybridization and array plate processing.
• Eight new plates of target are prepared on the Biomek FXP Target Prep Express.
Plate numbering scheme
• Target preparation on the Biomek FXP Target Prep Express: Sample Plates are numbered 1 through 8.
• Target prepared the previous week is processed on the GeneTitan MC Instrument. The plates denatured and loaded onto the GeneTitan™ MC Instrument are now referred to as plates A through H. Target prepared on the Biomek FXP Target Prep Express can be run on the GeneTitan MC Instrument in random order.
IMPORTANT! Experienced users and careful timing are critical for the successful execution of this workflow.
246 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 6 Processing 8 Axiom™ array plates per weekOverview of the 8-plate workflow 6
Week 1
Table 66 Overview of week 1: Target preparation on the Biomek FXP Target Prep Express
Day Activities Plates
1 • Amplify 8 plates of genomic DNA. 1 through 8
2 • Fragment and precipitate 3 plates amplified on Day 1.• Freeze 5 plates of amplified gDNA for fragmentation later in the week.
• 1, 5, 8• 2, 3, 4, 6, 7
3 • Fragment and precipitate 2 more amplified plates.• Centrifuge, dry, resuspend and QC the 3 plates precipitated on Day 2.
• 2, 3• 1, 5, 8
4 • Fragment and precipitate the 3 remaining amplified plates.• Centrifuge and dry the 2 plates precipitated on Day 3.
• 4, 6, 7• 2, 3
5 • Resuspend and QC the 2 plates dried on Day 4.• Centrifuge, dry, resuspend and QC the 3 plates precipitated on Day 4.
• 2, 3• 4, 6, 7
Table 67 Hands-on time required for target preparation on the Biomek FXP Target Prep Express per 96-array plate
Steps on the Biomek FXP Target Prep Express Time required
Amplification 30 minutes
Fragmentation 2 hours
Resuspension 45 minutes
Off-deck centrifugation and drying 75 minutes
Off-deck QC gel and OD 45 minutes
Denaturation only 30 minutes
Denaturation and GeneTitan reagent tray preparation 45 minutes
Transfer denatured samples to the GeneTitan MC Instrument 15 minutes
Transfer reagent trays to the GeneTitan MC Instrument 15 minutes
Total time required (includes setup) 7 hours total
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 247
Chapter 6 Processing 8 Axiom™ array plates per weekOverview of the 8-plate workflow6
Week 2
The hybridization time for the Axiom 2.0 Assay on the GeneTitan MC Instrument is 23.5–24 hours (Table 69). This provides a 30 minutes window during which you are prompted by the instrument control software to load the reagents required for washing and staining. We recommend that you begin loading the reagent trays onto the GeneTitan MC Instrument at the mid-point of this 30 minutes window. As such, the wash procedures will begin 24 hours after the start of hybridization. If catch-up is required in the framework of the 8-plate workflow, begin loading reagents at the beginning of this 30 minutes window (i.e., immediately after prompted by the software).
IMPORTANT! Maintaining consistent timing during the set up of the GeneTitan MC Instrument is critical to containing the user interventions of the 8 plate workflow within an 8 hour work day. Once a process begins late, there is little opportunity to catch up until the end of the workflow.
Table 68 Overview of week 2: Array processing in the GeneTitan™ MC Instrument
Day Activities Plates
1 • Hybridize 2 plates of denatured target • A and B
2 • Hybridize 2 plates of denatured target• Load reagent trays for fluidics and imaging of plates loaded on day 1
• C and D• A and B
3 • Hybridize 2 plates of denatured target• Load reagent trays for fluidics and imaging of plates loaded on day 2
• E and F• C and D
4 • Hybridize 2 plates of denatured target• Load reagent trays for fluidics and imaging of plates loaded on day 3
• G and H• E and F
5 • Load reagent trays for fluidics and imaging of plates loaded on day 4 • G and H
Table 69 Time required for Axiom array plate processing on the GeneTitan MC Instrument
Steps on the GeneTitan MC Instrument Time required
Hybridization of 2 plates• First plate loaded at 9:30 a.m.• Second plate loaded at 5:00 p.m.
23.5 hours each plate
Loading reagent trays that were prepared on the Biomek FXP Target Prep Express
15 minutes
Fluidics 5 hours each plate
Imaging 96 arrays: 7.5 hours
248 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 6 Processing 8 Axiom™ array plates per weekThawing frozen plates of amplified DNA 6
Thawing frozen plates of amplified DNA
To thaw frozen plates of amplified DNA:
1. Place the deep well plate in a small water bath.
For example, pour Millipore water into a small tray. Place the frozen plate on the water in the tray.
2. Leave the plate in the water bath for ~ 50 minutes until all wells have thawed.
3. Centrifuge at 1,000 rpm for 30 seconds.
4. To avoid cross-contamination of wells during vortexing:
a. Remove the seal and blot the top of the plate with a Kimwipes laboratory tissue.
b. Tightly reseal the plate with a fresh seal.
5. Vortex the plate for 30 seconds to thoroughly mix.
6. Centrifuge at 1,000 rpm for 30 seconds.
Automated target preparation for processing 8 Axiom™ array plates per week
Using 1 Biomek FXP Target Prep Express (Biomek workstation) and 1 GeneTitan MC Instrument, the Axiom™ 2.0 Genotyping Assay (using the 96-array plate) can be run at a throughput of 8 Axiom™ array plates per 5-day work week. This chapter includes tables that present the timing of the steps required to perform this workflow per 5-day work week, 8 hours per day.
During the initial week of startup, all work is done on the Biomek FXP Target Prep Express. You will process 8 plates of genomic DNA samples. At the end of this week, you will have what are now referred to as 8 plates of hyb-ready target (target). Subsequent weeks of the workflow involve the simultaneous processing of plates on the Biomek FXP Target Prep Express and on the GeneTitan MC Instrument. Each week:
• Eight plates of target from the previous week are denatured and transferred to the GeneTitan MC Instrument for hybridization and array plate processing.
• Eight new plates of target are prepared on the Biomek FXP Target Prep Express.
IMPORTANT! Experienced users and careful timing are critical for the successful execution of this workflow.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 249
Chapter 6 Processing 8 Axiom™ array plates per weekInitial target prep week—Biomek FXP Target Prep Express6
Initial target prep week—Biomek FXP Target Prep Express
Initial target prep week—Day 1
• Amplify 8 plates of gDNA samples.
IMPORTANT! • All amplifications should be set up on Day 1 to allow for a 23 1 hour
amplification incubation for each plate.
• Begin thawing the amplification reagents, particularly the Axiom 2.0 Amp Soln, 60 minutes prior to the start of each reaction.
Monday a.m. Monday p.m.
8 9 10 11 12 1 2 3 4 5
1
2
3
4
5
6
7
8
Figure 137 Initial target prep week—Day 1 activities
Table 70 Initial target prep week—Day 1 activities
Activity Plate number
Instrument Approximate start times
DNA Amplification 1
Biomek FXP Target Prep Express
10:00 a.m.
DNA Amplification 2 10:30 a.m.
DNA Amplification 3 11:00 a.m.
DNA Amplification 4 11:30 a.m.
DNA Amplification 5 12:00 p.m.
DNA Amplification 6 1:00 p.m.
DNA Amplification 7 1:30 p.m.
DNA Amplification 8 2:00 p.m.
250 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 6 Processing 8 Axiom™ array plates per weekInitial target prep week—Biomek FXP Target Prep Express 6
Initial target prep week—Day 2
• Fragment and precipitate plates 1, 5 and 8.
• Freeze amplified plates 2, 3, 4, 6, and 7 after each plate has incubated for 23 hours.
IMPORTANT! • Plates 1, 5 and 8 are fragmented and precipitated on Day 2 without freezing to
preserve a 23 hours amplification incubation.
• Store all plates not fragmented and precipitated on Day 2 at –20°C following 23 hours of amplification reaction incubation.
• Precipitation is carried out at –20°C overnight.
Tuesday a.m. Tuesday p.m.
8 9 10 11 12 1 2 3 4 5
1—Fragment/Precipitate
s2
s3
s4
5—Fragment/Precipitate
s6
s7
8—Fragment/Precipitate
s = Seal tightly and store at –20°C.
Figure 138 Initial target prep week—Day 2 activities
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 251
Chapter 6 Processing 8 Axiom™ array plates per weekInitial target prep week—Biomek FXP Target Prep Express6
Initial target prep week—Day 3
• Thaw plates 2 and 3 (see ʺThawing frozen plates of amplified DNAʺ on page 249).
• Fragment and precipitate plates 2 and 3.
• Centrifuge, dry, resuspend and QC plates 1, 5 and 8.
Table 71 Initial target prep week—Day 2 activities
Activity Plate number
Instrument Approximate start times
Fragment and precipitate 1 Biomek FXP Target Prep Express 9:30 a.m.
Freeze (–20°C) 2 — 10:00 a.m.
Freeze (–20°C) 3 — 10:30 a.m.
Freeze (–20°C) 4 — 11:00 a.m.
Fragment and precipitate 5 Biomek FXP Target Prep Express 11:30 a.m.
Freeze (–20°C) 6 — 12:30 p.m.
Freeze (–20°C) 7 — 1:00 p.m.
Fragment and precipitate 8 Biomek FXP Target Prep Express 1:30 p.m.
IMPORTANT! • Amplified plates that are frozen must be thawed and thoroughly mixed by
following the procedure under ʺThawing frozen plates of amplified DNAʺ on page 249.
• After being centrifuged and dried, plates 1, 5 and 8 are sealed and placed in a 4°C refrigerator until further processing later the same day.
• Precipitation is carried out at –20°C overnight.
• Prior to resuspension and QC, plates 1, 5 and 8 must be brought to room temperature (place on benchtop for 30 minutes).
252 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 6 Processing 8 Axiom™ array plates per weekInitial target prep week—Biomek FXP Target Prep Express 6
Wednesday a.m. Wednesday p.m.
8 9 10 11 12 1 2 3 4 5
2 - RT
2—Fragment/Precipitate
1, 5, 8Centrif/Dry
3 - RT
3—Fragment/Precipitate
1-RT 1-Resus
1-QC
5-RT 5-Resus
5-QC
8-RT 8-Resus
8-QC
RT = Bring plate to room temperatureCentrif = centrifuge offlineResus = resuspendQC = fragmentation QC gel and OD quantitation
Figure 139 Initial target prep week—Day 3 activities
Table 72 Initial target prep week—Day 3 activities
Activity Plate number
Instrument Approximate start times
Bring plate to room temperature (RT) 2 — 8:30 a.m.
Fragment and precipitate 2 Biomek FXP Target Prep Express 9:30 a.m.
Centrifuge and dry 1, 5, 8 Plate centrifuge and oven 9:45 a.m.
Bring plate to room temperature (RT) 3 — 10:30 a.m.
Fragment and precipitate 3 Biomek FXP Target Prep Express 11:30 a.m.
Resuspension 1 Biomek FXP Target Prep Express, 1:30 p.m.
Off-deck QC 1 Plate spectrophotometer, e-gel system 2:15 p.m.
Resuspension 5 Biomek FXP Target Prep Express 2:15 p.m.
Off-deck QC 5 Plate spectrophotometer, e-gel system 3:00 p.m.
Resuspension 8 Biomek FXP Target Prep Express 3:00 p.m.
Off-deck QC 8 Plate spectrophotometer, e-gel system 3:45 p.m.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 253
Chapter 6 Processing 8 Axiom™ array plates per weekInitial target prep week—Biomek FXP Target Prep Express6
Initial target prep week—Day 4
• Fragment and precipitate plates 4, 6 and 7.
• Centrifuge and dry plates 2 and 3.
IMPORTANT! • Amplified plates that are frozen must be thawed and thoroughly mixed by
following the procedure under ʺThawing frozen plates of amplified DNAʺ on page 249.
• Precipitation is carried out at –20°C overnight.
• After being centrifuged and dried, plates 2 and 3 are sealed and stored at –20°C.
Thursday a.m. Thursday p.m.
8 9 10 11 12 1 2 3 4 5
4 - RT
4—Fragment/Precipitate
2, 3Centrif/Dry
6 - RT
6—Fragment/Precipitate
7 - RT
7—Fragment/Precipitate
RT = Bring plate to room temperatureCentrif = Centrifuge
Figure 140 Initial target prep week—Day 4 activities
Table 73 Initial target prep week—Day 4 activities
Activity Plate number Instrument Approximate start times
Thaw 4 — 8:30 a.m.
Fragment and precipitate 4 Biomek FXP Target Prep Express 9:30 a.m.
Centrifuge and dry 2, 3 Plate centrifuge and oven 9:45 a.m.
Thaw 6 — 10:30 a.m.
Fragment and precipitate 6 Biomek FXP Target Prep Express 11:30 a.m.
Thaw 7 — 12:30 p.m.
Fragment and precipitate 7 Biomek FXP Target Prep Express 1:30 p.m.
254 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 6 Processing 8 Axiom™ array plates per weekInitial target prep week—Biomek FXP Target Prep Express 6
Initial target prep week—Day 5
• Centrifuge and dry plates 4, 6 and 7.
• Resuspend and QC plates 2, 3, 4, 6 and 7.
IMPORTANT! • Plates 2 and 3 must be brought to room temperature for 90 minutes prior to
resuspension.
• After being centrifuged and dried, plates 4, 6 and 7 are sealed. Place plates 6 and 7 in a 4°C refrigerator until further processing later the same day. Plate 4 can be left on the benchtop.
• Prior to resuspension and QC, plates 6 and 7 must be brought to room temperature (place on benchtop for 30 minutes).
Friday a.m. Friday p.m.
8 9 10 11 12 1 2 3 4 5
2 - RT
3 - RT
2-Resus
2-QC
4, 6, 7Centrif/Dry
3-Resus
3-QC
4-Resus
4-QC
6-RT 6-Resus
6-QC
7-RT 7-Resus
7-QC
RT = Bring plate to room temperature.Resus = ResuspensionQC = fragmentation QC gel and OD quantitation
Figure 141 Initial target prep week—Day 5 activities
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 255
Chapter 6 Processing 8 Axiom™ array plates per weekInitial target prep week—Biomek FXP Target Prep Express6
Table 74 Initial target prep week—day 5 activities
Activity Plate number Instrument Approximate start times
Bring to room temperature 2 — 8:00 a.m.
Bring to room temperature 3 — 8:45 a.m.
Resuspension and QC 2 Biomek FXP Target Prep Express 9:30 a.m.
Centrifuge and dry 4, 6, 7 Plate centrifuge and oven 9:45 a.m.
Resuspension and QC 3
Biomek FXP Target Prep Express
10:15 a.m.
Resuspension and QC 4 11:00 a.m.
Resuspension and QC 6 1:15 p.m.
Resuspension and QC 7 2:00 p.m.
256 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 6 Processing 8 Axiom™ array plates per weekSimultaneous 8-plate workflow 6
Simultaneous 8-plate workflow
The tables on the following pages provide a breakdown of the timing involved to simultaneously:
• GeneTitan MC Instrument: Process 8 array plates per week with target prepared the previous week on the Biomek FXP Target Prep Express.
• Biomek FXP Target Prep Express: Prepare 8 new plates of target for processing the following week on the GeneTitan MC Instrument.
Eight-plate workflow—Day 1
Biomek FXP Target Prep Express activities• Denature 2 plates of target prepared the previous week (Plates A and B).
• Amplify 8 new plates of genomic DNA (1 through 8)
GeneTitan MC Instrument activities• Transfer denatured plates to the GeneTitan MC Instrument and begin
hybridization (Plates A and B).
IMPORTANT! Timing is critical.
• The Biomek FXP Target Prep Express will be in operation for a large percentage of each work day.
• Plates for target preparation on the Biomek FXP Target Prep Express are numbered 1 through 8.
• Plates with target that are now ready for denaturation and transfer to the GeneTitan MC Instrument for hybridization, fluidics processing, and imaging are lettered A through H.
• Plates of target from the previous week (1–8) can be processed on the GeneTitan MC Instrument in any order.
IMPORTANT! • Plates 1 through 8 are referred to as A through H when denatured and loaded onto
the GeneTitan MC Instrument. Plates 1 through 8 can go onto the GeneTitan MC Instrument in any order.
• All amplifications are set up on Day 1 to maintain a 23 1 hour amplification incubation for each plate.
• Begin thawing the amplification reagents, particularly the Axiom 2.0 Amp Soln, 60 minutes prior to the start of each reaction.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 257
Chapter 6 Processing 8 Axiom™ array plates per weekSimultaneous 8-plate workflow6
Monday a.m. Monday p.m.
8 9 10 11 12 1 2 3 4 5
Den A Hyb A
1
2
3
4
5
6
7
8
Den B Hyb B
• Den = denature• Den time period includes 15 minutes to transfer the denatured Sample Plate from the Biomek workstation to the
GeneTitan MC Instrument.• Begin thawing the amplification reagents, particularly the Axiom 2.0 Amp Soln, 60 minutes prior to the start of each
reaction.
Figure 142 Eight-plate workflow—Day 1 activities
Table 75 Eight-plate workflow—Day 1 activities
Activity Plate number Instrument Approximate start times
Denature on the FX A Biomek FXP Target Prep Express 8:45 a.m.
Load onto the GT and hybridize A GeneTitan MC Instrument 9:30 a.m.
DNA Amplification 1
Biomek FXP Target Prep Express
10:00 a.m.
DNA Amplification 2 10:30 a.m.
DNA Amplification 3 11:00 a.m.
DNA Amplification 4 11:30 a.m.
DNA Amplification 5
Biomek FXP Target Prep Express
12:00 p.m.
DNA Amplification 6 1:00 p.m.
DNA Amplification 7 1:30 p.m.
DNA Amplification 8 2:00 p.m.
Denature on the FX B Biomek FXP Target Prep Express 4:15 p.m.
Load onto the GT and hybridize B GeneTitan MC Instrument 5:00 p.m.
258 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 6 Processing 8 Axiom™ array plates per weekSimultaneous 8-plate workflow 6
Eight-plate workflow—Day 2
Biomek FXP Target Prep Express activities• Denature 2 plates of target from the previous week (plates C and D).
• Prepare reagent trays for the GeneTitan MC Instrument (for Plates A and B already on the GeneTitan MC Instrument).
• Transfer the denatured samples and reagent trays to the GeneTitan MC Instrument.
• Fragment and precipitate plates 1, 5 and 8.
• Freeze amplified plates 2, 3, 4, 6, and 7 after each plate has incubated for 23 hours
GeneTitan MC Instrument activities• Load reagent trays for A and B. These plates are moved from the hybridization
oven to the fluidics area. After fluidics, the plates move to the imaging area of the instrument. Load Plates C and D for hybridization.
IMPORTANT! • Plates 1, 5 and 8 are fragmented and precipitated without freezing to preserve a
23 hours amplification incubation.
• All plates not fragmented and precipitated on Day 2 should be stored at –20°C following 23 hr of amplification reaction incubation.
• Precipitation is carried out at –20°C overnight.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 259
Chapter 6 Processing 8 Axiom™ array plates per weekSimultaneous 8-plate workflow6
Tuesday a.m. Tuesday p.m.
8 9 10 11 12 1 2 3 4 5
Thaw GT
rgntsDen C Hyb C
End Hyb
A
GT rgnt plates for A Fluidics and Scan A
1—Fragment/Precipitate
s2
s3
s4
5—Fragment/Precipitate
s6
s7
8—Fragment/Precipitate
Thaw GT
rgntsDen D
Hyb D
Hyb B23.5–24 hours
GT rgnt plates for B
**B
** B = Fluidics and Scan B
Den = denature
Denature and GT rgnt plate time periods include 15 minutes to transfer the denatured plate and reagent trays from the Biomek to the GeneTitan MC Instrument.
s = Seal tightly and store at –20°C.
GT rgnt plates = reagent trays for GeneTitan array plate processing.
Figure 143 Eight-plate workflow—Day 2 activities
260 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 6 Processing 8 Axiom™ array plates per weekSimultaneous 8-plate workflow 6
Table 76 Eight-plate workflow—Day 2 activities
Activity Plate designation
Instrument Approximate start times
Thaw reagent for GT rgnt plate preparation
A — 8:00 a.m.
Concurrently:• Denature C• Prepare GT rgnt plates for A• Transfer to the GeneTitan MC
Instrument
Denature CReagents for A
Biomek FXP Target Prep Express 8:30 a.m.
• Load GT rgnt plates and begin fluidics processing for A
• Load C and begin hybridization
A to fluidicsC to hybridization
oven
GeneTitan MC Instrument 9:30 a.m.
Fragment and precipitate 1 Biomek FXP Target Prep Express 9:30 a.m.
Freeze (–20°C) 2 — 10:00 a.m.
Freeze (–20°C) 3 — 10:30 a.m.
Freeze (–20°C) 4 — 11:00 a.m.
Fragment and precipitate 5 Biomek FXP Target Prep Express 11:30 a.m.
Freeze (–20°C) 6 — 12:30 p.m.
Freeze (–20°C) 7 — 1:00 p.m.
Fragment and precipitate 8 Biomek FXP Target Prep Express 1:30 p.m.
Thaw reagent for GT rgnt plate preparation
B — 3:30 p.m.
Concurrently:• Denature D• Prepare GT rgnt plates for B• Transfer to the GeneTitan MC
Instrument
Denature DReagents for B
Biomek FXP Target Prep Express 4:00 p.m.
• Load GT rgnt plates and begin fluidics processing for B
• Load D and begin hybridization
B to fluidicsD to hybridization
oven
GeneTitan MC Instrument 5:00 p.m.
Denature and GT rgnt plate preparation time periods include 15 minutes to transfer the denatured plate and reagent trays from the Biomek to the GeneTitan MC Instrument.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 261
Chapter 6 Processing 8 Axiom™ array plates per weekSimultaneous 8-plate workflow6
Eight-plate workflow—Day 3
Off-deck activities• Thaw plates 2 and 3.
• QC—OD quantitation and fragmentation gel for Plates 1, 5, 8.
Biomek FXP Target Prep Express activities• Denature 2 plates of target from the previous week (Plates E and F).
• Prepare reagent trays for the GeneTitan MC Instrument (for Plates C and D already on the GeneTitan MC Instrument).
• Transfer the denatured samples and reagent trays to the GeneTitan MC Instrument.
• Fragment and precipitate Plates 2 and 3.
• Centrifuge, dry, resuspend and QC Plates 1, 5, 8.
GeneTitan MC Instrument activities• Load reagent trays for Plates C and D. These plates are moved from the
hybridization oven to the fluidics area. After fluidics, the plates will move to the imaging area of the instrument. Also load Plates E and F for hybridization.
262 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 6 Processing 8 Axiom™ array plates per weekSimultaneous 8-plate workflow 6
Wednesday a.m. Wednesday p.m.
8 9 10 11 12 1 2 3 4 5
Thaw GT
rgntsDen E Hyb E
End Hyb C
GT rgnt plates for C
Fluidics and Scan C
2 – RT
2—Fragment/Precipitate
1, 5, 8Centrif/Dry
3 - RT
3—Fragment/Precipitate
1-RT 1-Resus 1-QC
5-RT 5-Resus 5-QC
8-RT 8-Resus 8-QC
Thaw GT
rgntsDen F Hyb F
Hyb DGT rgnt
plates for DFluidics
and Scan D
RT = Bring plate to room temperatureCentrif = centrifugeResus = resuspendQC = fragmentation QC gel and OD quantitationGT rgnt plates = reagent trays for GeneTitan array plate processing• Den time period includes 15 minutes to transfer the denatured Sample Plate and reagent trays from the Biomek to the
GeneTitan MC Instrument
Figure 144 Eight-plate workflow—Day 3 activities
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 263
Chapter 6 Processing 8 Axiom™ array plates per weekSimultaneous 8-plate workflow6
Table 77 Eight-plate workflow—Day 3 activities
Activity Plate number Instrument Approximate start times
Thaw reagents for GT rgnt plate preparation
C — 8:00 a.m.
Bring plate to room temperature (RT) 2 — 8:30 a.m.
Concurrently:• Denature E• Prepare GT rgnt plates for C• Transfer to the GeneTitan MC Instrument
• Denature E• Reagents for C
Biomek FXP Target Prep Express 8:30 a.m.
• Load GT rgnt plates and begin fluidics processing for C
• Load E and begin hybridization
• C to fluidics
• E to hyb oven
GeneTitan MC Instrument 9:30 a.m.
Fragment and precipitate 2 Biomek FXP Target Prep Express 9:30 a.m.
Centrifuge and dry 1, 5, 8 Plate centrifuge and oven 9:45 a.m.
Bring plate to room temperature (RT) 3 — 10:30 a.m.
Fragment and precipitate 3 Biomek FXP Target Prep Express 11:30 a.m.
Resuspension 1 Biomek FXP Target Prep Express 1:30 p.m.
Off-deck QC 1 Plate spectrophotometer, e-gel system 2:15 p.m.
Resuspension 5 Biomek FXP Target Prep Express 2:15 p.m.
Off-deck QC 5 Plate spectrophotometer, e-gel system 3:00 p.m.
Resuspension 8 Biomek FXP Target Prep Express 3:00 p.m.
Thaw reagents for GT rgnt plate preparation
F — 3:30 p.m.
Off-deck QC 8 Plate spectrophotometer, e-gel system 3:45 p.m.
Concurrently:• Denature F• Prepare GT rgnt plates for D• Transfer to the GeneTitan MC Instrument
• Denature F• Reagents for D
Biomek FXP Target Prep Express 4:00 p.m.
• Load GT rgnt plates and begin fluidics processing for D
• Load F and begin hybridization
• D to fluidics
• F to hyb oven
GeneTitan MC Instrument 5:00 p.m.
264 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 6 Processing 8 Axiom™ array plates per weekSimultaneous 8-plate workflow 6
Eight-plate workflow—Day 4
Off-Deck activitiesBring Plates 4, 6 and 7 to room temperature.
Biomek FXP Target Prep Express activities• Denature 2 plates of target from the previous week (Plates G and H).
• Prepare reagent trays for the GeneTitan MC Instrument (for Plates E and F already on the GeneTitan MC Instrument).
• Transfer the denatured samples and reagent trays to the GeneTitan MC Instrument.
• Fragment and precipitate Plates 4, 6, 7.
• Centrifuge and dry Plates 2 and 3.
GeneTitan MC Instrument activities• Load reagent trays for E and F. These plates are moved from the hybridization
oven to the fluidics area. After fluidics, the plates move to the imaging area of the instrument. Load Plates G and H for hybridization.
IMPORTANT! • Amplified plates that are frozen must be thawed and thoroughly mixed by
following the procedure under ʺThawing frozen plates of amplified DNAʺ on page 249.
• Precipitation is carried out at –20°C overnight.
• After being centrifuged and dried, Plates 2 and 3 are sealed and stored at –20°C.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 265
Chapter 6 Processing 8 Axiom™ array plates per weekSimultaneous 8-plate workflow6
Thursday a.m. Thursday p.m.
8 9 10 11 12 1 2 3 4 5
Thaw GT
rgntsDen G Hyb G
End Hyb E
GT rgnt plates for E
Fluidics and Scan E
4 - RT
4—Fragment/Precipitate
2, 3 Centrif/Dry
6 - RT
6—Fragment/Precipitate
7 - RT
7—Fragment/Precipitate
Thaw GT
rgntsDen H Hyb H
Hyb FGT rgnt
plates for FFluidics
and Scan F
RT = Bring plate to room temperatureCentrif = centrifugeDenature and GT rgnt plate preparation time periods include 15 minutes to transfer the denatured plate and reagent trays from the Biomek to the GeneTitan MC Instrument.
Figure 145 Eight-plate workflow— Day 4 Activities
266 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 6 Processing 8 Axiom™ array plates per weekSimultaneous 8-plate workflow 6
Table 78 Eight-plate workflow— Day 4 activities
Activity Plate designation
Instrument Approximate start times
Thaw reagents for GT rgnt plate preparation
E — 8:00 a.m.
Bring plate to room temperature (RT) 4 — 8:30 a.m.
Concurrently:• Denature G• Prepare GT rgnt plates for E• Transfer to the GeneTitan MC Instrument
• Denature D• Reagent for E
Biomek FXP Target Prep Express 8:30 a.m.
• Load GT rgnt plates and begin fluidics processing for E
• Load G and begin hybridization
• E to fluidics
• G to hyb oven
GeneTitan MC Instrument 9:30 a.m.
Fragment and precipitate 4 Biomek FXP Target Prep Express 9:30 a.m.
Centrifuge and dry 2, 3 Plate centrifuge and oven 9:45 a.m.
Bring plate to room temperature (RT) 6 — 10:30 a.m.
Fragment and precipitate 6 Biomek FXP Target Prep Express 11:30 a.m.
Bring plate to room temperature (RT) 7 — 12:30 a.m.
Fragment and precipitate 7 Biomek FXP Target Prep Express 1:30 a.m.
Thaw reagents for GT rgnt plate preparation
F — 3:30 p.m.
Concurrently:• Denature H• Prepare GT rgnt plates for F• Transfer to the GeneTitan MC Instrument
• Denature H • Reagents for F
Biomek FXP Target Prep Express 4:00 p.m.
• Load GT rgnt plates and begin fluidics processing for F
• Load H and begin hybridization
• F to fluidics
• H to hyb oven
GeneTitan MC Instrument 5:00 p.m.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 267
Chapter 6 Processing 8 Axiom™ array plates per weekSimultaneous 8-plate workflow6
Eight-plate workflow—Day 5
Off-Deck activitiesBring Plates 2 and 3 to room temperature.
Biomek FXP Target Prep Express activities• Centrifuge and dry Plates 4, 6, 7.
• Resuspend and QC Plates 2, 3, 4, 6, 7.
GeneTitan MC Instrument activitiesLoad reagent trays for plates E and F. These plates are moved from the hybridization oven to the fluidics area. After fluidics, the plates will move to the imaging area of the instrument. Also load Plates G and H for hybridization.
IMPORTANT! • Plates 2 and 3 must be brought to room temperature for 90 minutes prior to
resuspension.
• After being centrifuged and dried, plates 4, 6 and 7 are sealed. Place Plates 6 and 7 in a 4°C refrigerator until further processing later the same day. Plate 4 can be left on the benchtop.
• Prior to resuspension and QC, Plates 6 and 7 must be brought to room temperature (place on benchtop for 30 minutes).
268 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 6 Processing 8 Axiom™ array plates per weekSimultaneous 8-plate workflow 6
Friday a.m. Friday p.m.
8 9 10 11 12 1 2 3 4 5
End Hyb G
GT rgnt plates for G
Fluidics and Scan G
2 - RT
3 - RT
2-Resus 2-QC
4, 6, 7Centrif/Dry
3-Resus 3-QC
4-Resus 4-QC
6-RT 6-Resus 6-QC
7-RT 7-Resus 7-QC
Hyb HRgnt Plate
Prep HFluidics
and Scan H
RT = Bring plate to room temperature.Resus = ResuspensionQC = fragmentation QC gel and OD quantitationGT rgnt plate preparation time periods include 15 minutes to transfer the denatured plate and reagent trays from the Biomek to the GeneTitan MC Instrument.
Figure 146 Eight-plate workflow—Day 5 activities
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 269
Chapter 6 Processing 8 Axiom™ array plates per weekSimultaneous 8-plate workflow6
Table 79 Eight-plate workflow—Day 5 activities
Activity Plate number Instrument Approximate start times
Bring to room temperature 2 — 8:00 a.m.
Prepare GeneTitan reagent trays
G Biomek FXP Target Prep Express 8:30 a.m.
Load reagent trays and begin fluidics processing for G
G GeneTitan MC Instrument 9:30 a.m.
Bring to room temperature 3 — 8:45 a.m.
Resuspension and QC 2 Biomek FXP Target Prep Express 9:30 a.m.
Centrifuge and dry 4, 6, 7 Plate centrifuge and oven 9:45 a.m.
Resuspension and QC 3
Biomek FXP Target Prep Express
10:15 a.m.
Resuspension and QC 4 11:00 a.m.
Resuspension and QC 6 1:15 p.m.
Resuspension and QC 7 2:00 p.m.
Prepare GeneTitan reagent trays
H Biomek FXP Target Prep Express 4:00 p.m.
Load reagent trays and begin fluidics processing for H
H GeneTitan MC Instrument 5:00 p.m.
270 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
7 Processing 3 Axiom™ array platesper week
The 3 array plate/week automated target prep workflow enables you to do the target preparation and array processing for 3 Axiom array plates in the same week.
This workflow is performed using 1 Biomek FXP Target Prep Express (Biomek workstation) (see Chapter 3, ʺTarget preparation on the Biomek FXP with Windows® XPʺ on page 22) and 1 GeneTitan MC Instrument (see Chapter 5, ʺArray processing with the GeneTitan™ MC Instrumentʺ on page 209). This chapter includes tables that present the timing of the steps required to perform this workflow per 5-day work week, 8 hours per day.
The 3 plate per week automated workflow is described in the following sections:
• ʺOverview of the 3-plate workflow for automated target preparationʺ
• ʺThawing frozen plates of amplified DNAʺ on page 274
• ʺTarget prep and array processingʺ on page 275
IMPORTANT! Experienced users and careful timing are critical for the successful execution of this workflow.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 271
Chapter 7 Processing 3 Axiom™ array plates per weekOverview of the 3-plate workflow for automated target preparation7
Overview of the 3-plate workflow for automated target preparation
The table below displays the timing and duration of the hands-on processing necessary for performing the 3-plate workflow by 1 person.
The 3 plates are referred to as Plates A, B and C in the target prep and in the GeneTitan Array Processing.
In order to process 3 plates during a 40-hour week, the steps should be performed in the order and with the timing described in this chapter.
Figure 147 Three plate per week automated target prep workflow
PlateDay 1 Day 2 Day 3 Day 4 Day 5
AM PM AM PM AM PM AM PM AM PM8 9 10 11 12 1 2 3 4 5 6 8 9 10 11 12 1 2 3 4 5 6 8 9 10 11 12 1 2 3 4 5 6 8 9 10 11 12 1 2 3 4 5 6 8 9 10 11 12 1 2 3 4 5 6
A
B
C
NotesBegin thawing required reagents for the process.Begin warming Axiom array plate to room temperature
x Day 2 11 AM: Freeze Plate Cy Day 3 Noon: start plate C thawingz Day 4 5:00 PM: Coupled operations on GeneTitan MC Instrument: Load reagents for Plate A and hybridization tray
and array plate for Plate CColor Code
Amplification Fragmentation and Precipitation Centrifugation and Drying Resuspension and Hybridization Mix Prep QC Sample Denature/load array plate and hybridization tray into the GeneTitan MC Instrument GeneTitan MC reagent trays prep and loading
x y z
z
272 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 7 Processing 3 Axiom™ array plates per weekOverview of the 3-plate workflow for automated target preparation 7
Table 80 Daily steps for automated target prep workflow for 3 plates/week
Day Activities
1 Amplify 3 plates of genomic DNA
2 1. Fragment and precipitate 2 plates amplified on Day 12. Freeze Plate C at –20°C
3 1. Centrifuge and dry Plate A and Plate B2. Resuspend and QC Plate A3. Resuspend and QC Plate B4. Denature Plate A and load samples and array plate into the GeneTitan MC
Instrument5. Store Plate B at –20°C6. Thaw Plate C frozen on Day 27. Fragment and precipitate Plate C amplified on Day 1
4 1. Denature Plate B and load samples and array plate into the GeneTitan MC Instrument
2. Centrifuge, dry, resuspend, and QC Plate C3. Prepare GeneTitan reagent trays for Plate A and load into the GeneTitan
MC Instrument and denature Plate C
5 1. Prepare GeneTitan reagent trays for Plate B and load into the GeneTitan MC Instrument
2. Prepare GeneTitan reagent trays for Plate C and load into the GeneTitan MC Instrument
Table 81 Time Required for Target Preparation on the Biomek FXP Target Prep Express per 96-Array Plate
Steps on the Biomek FXP Target Prep Express Time required
Amplification 30 minutes
Fragmentation 2 hours
Resuspension 45 minutes
Off-deck centrifugation and drying 75 minutes
Off-deck QC gel and OD 45 minutes
Denaturation only 30 minutes
Transfer denatured samples to the GeneTitan MC Instrument 15 minutes
Prepare and Load reagent trays to the GeneTitan MC Instrument 60 minutes
Total Time Required includes deck setup. 7 hours
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 273
Chapter 7 Processing 3 Axiom™ array plates per weekThawing frozen plates of amplified DNA7
Thawing frozen plates of amplified DNA
The automated 3 plate workflow described in this chapter requires freezing Plate C of DNA following amplification on Day 2. You should thaw Plate C prior to the fragmentation step on Day 2.
To thaw frozen plates of amplified DNA:
1. Place the deep well plate in a small water bath.
For example, pour Millipore water into a small tray. Place the frozen plate on the water in the tray.
2. Leave the plate in the water bath for ~50 minutes until all wells have thawed.
3. Centrifuge at 1,000 rpm for 30 seconds.
4. To avoid cross-contamination of wells during vortexing:
a. Remove the seal and blot the top of the plate with a Kimwipes laboratory tissue.
b. Tightly reseal the plate with a fresh seal.
5. Vortex the plate for 30 seconds to thoroughly mix.
6. Centrifuge at 1,000 rpm for 30 seconds.
274 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 7 Processing 3 Axiom™ array plates per weekTarget prep and array processing 7
Target prep and array processing
The tables in the sections below show the steps that need to be performed on each day of the workflow.
Three plate/week workflow—Day 1
Note: Genomic DNA sample for amplification should be prepared as described in ʺGenomic DNA preparation and requirementsʺ on page 13.
See ʺStage 1: DNA Amplificationʺ on page 51 for more information on the protocol.
Figure 148 Three plate/week automated target prep workflow—Day 1 activities
Plate
Day 1 a.m. Day 1 p.m.
8 9 10 11 12 1 2 3 4 5
A Amp
B Amp
C Amp
Notes
Begin thawing reagents and materials for the process
Color Code
Amp Amplification (see "Stage 1: DNA Amplification" on page 51)
Table 82 Three plate/week auto target prep workflow—Day 1
Time Topic
9:30 Prepare DNA amplification reagents for Plate A
10:30 Prepare DNA amplification reagents for Plate C
10:30 Start DNA amplification Plate A
11:30 Start DNA amplification Plate C
1:30 Prepare DNA amplification reagents for Plate B
2:30 Start DNA amplification Plate B
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 275
Chapter 7 Processing 3 Axiom™ array plates per weekTarget prep and array processing7
Three plate/week workflow—Day 2
Figure 149 Three plate/week workflow—Day 2 activities
Plate
Day 2 a.m. Day 2 p.m.
8 9 10 11 12 1 2 3 4 5
A Frag
B Frag
C
Notes
Begin thawing required reagents
X Freeze Plate C
Color Code
Frag Fragmentation and precipitation (see "Stage 2: Fragmentation and Purification" on page 65)
X
Table 83 Three plate/week auto target prep workflow—Day 2
Time Topic
9:30 Prepare Frag reagents for Plate A
10:00 Start Fragmentation Plate A
11:00 Transfer Amplified Plate C to –20°C
1:30 Prepare Frag reagents for Plate B
2:00 Start Fragmentation Plate B
276 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 7 Processing 3 Axiom™ array plates per weekTarget prep and array processing 7
Three plate/week workflow—Day 3
Figure 150 Three plate/week automated target prep workflow—Day 3 activities
Plate
Day 3 a.m. Day 3 p.m.
8 9 10 11 12 1 2 3 4 5
A Cent/Dry R/HP QC Denat/ hyb
B Cent/Dry R/HP QC
C Frag
Notes
Begin thawing required reagents
Begin warming Axiom array plate to room temperature
X Store Hyb Ready Plate A and Plate B at –20°C
Y Begin thawing Amp Plate C
Color Codes
Frag Fragmentation and Precipitation (see "Stage 2: Fragmentation and Purification" on page 65)
Cent/Dry Centrifugation and Drying (see "5. Centrifuge and dry pellets" on page 75)
R/HP Resuspension and Hyb Mix Prep (see "Stage 3: Resuspension and hybridization preparation" on page 76)
QC Quality control checks
Denat/hyb
Sample Denature/load array plate and hybridization tray into the GeneTitan MC Instrument (see "Stage 4: Preparation for the GeneTitan™ MC Instrument" on page 89)
X
Y
X
Table 84 Three plate/week automated target prep workflow—Day 3
Time Topic
9:00 Start Centrifugation of Plate A and Plate B.
9:40 Start Drying of Plate A and Plate B.
10:05 Start Resuspension and Hybridization Preparation Plate A, keep Plate B sealed at room temperature.
10:45 Start Resuspension and Hybridization Preparation Plate B.
11:30 Run QC on Plate A and Plate B.
12:00 Transfer Hyb-ready Plate A and Plate B to freezer (–20°C).Thaw Amplified Plate C.
12:30 Prepare fragmentation reagents for Plate C.
1:00 Start fragmentation of Plate C.
4:30 Start denature Sample Plate A
5:00 Start Hyb-Wash-Scan run Plate A.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 277
Chapter 7 Processing 3 Axiom™ array plates per weekTarget prep and array processing7
Three plate/week workflow—Day 4 IMPORTANT! The GeneTitan reagent trays for array processing cannot be loaded
until the array plate has finished hybridization, and they should not be prepared more than 1.5 hours before hybridization will finish. The GeneTitan reagent trays cannot be prepared ahead of time and stored.
Figure 151 Three plate/week automated target prep workflow—Day 4
Plate
Day 4 a.m. Day 4 p.m.
8 9 10 11 12 1 2 3 4 5
A
GT Reagent
Prep/Load
B Denat/hyb
C Cent/Dry R/HP QCDenat/hyb
Notes
Begin thawing required reagents
Begin warming Axiom array plate to room temperature
X Store Hyb Ready Plate C at –20°C
Z Coupled GeneTitan Instrument operations: Load reagent trays for Plate A and hyb tray/array plate for Plate C.
Color Codes
Cent/Dry Centrifugation and Drying (See "5. Centrifuge and dry pellets" on page 75)
R/HP Resuspension and Hyb Mix Prep (see "Stage 3: Resuspension and hybridization preparation" on page 76)
QC Quality Control Checks
Denat/Hyb Sample Denature/load array plate and hybridization tray into the GeneTitan MC Instrument (see "Stage 4: Preparation for the GeneTitan™ MC Instrument" on page 89)
GT Reagent prep/load
GeneTitan reagent trays prep and load (see "Stage 4: Preparation for the GeneTitan™ MC Instrument" on page 89)
ZX
Z
278 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 7 Processing 3 Axiom™ array plates per weekTarget prep and array processing 7
Table 85 Three Plate/Week Auto Target Prep Workflow—Day 4
Time Topic
9:00 Start Denature Sample for Plate B (see "Stage 4: Preparation for the GeneTitan™ MC Instrument" on page 89)
9:30 Start Hyb-Wash-Scan run for Plate B
9:30 Start centrifugation of Plate C
10:10 Start drying of Plate C
10:35 Start resuspension and hybridization preparation Plate C
11:10 Run QC on Plate C
11:40 Transfer Plate C to freezer (–20°C)
3:30 Prepare wash and stain reagents for Plate A
4:00 Prepare GeneTitan reagent trays for Plate A (see "Stage 4: Preparation for the GeneTitan™ MC Instrument" on page 89)
4:30 Start denature samples for Plate C
5:00 Load GeneTitan reagent trays for Plate A
5:00 Start Hyb-Wash-Scan run for Plate C
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 279
Chapter 7 Processing 3 Axiom™ array plates per weekTarget prep and array processing7
Three plate/week workflow—Day 5 IMPORTANT! The GeneTitan reagent trays for array processing cannot be loaded
until the array plate has finished hybridization, and they should not be prepared more than 1.5 hours before hybridization will finish. The GeneTitan reagent trays cannot be prepared ahead of time and stored.
Figure 152 Three plate/week automated target prep workflow—Day 5
Plate
Day 5 a.m. Day 5 p.m.
8 9 10 11 12 1 2 3 4 5
A
B
GT Reagent
Prep/Load
C
GT Reagent
Prep/Load
Notes
Begin thawing required reagents
Color Codes
GT Reagent
Prep/Load
GeneTitan reagent trays prep and load (see "Stage 4: Preparation for the GeneTitan™ MC Instrument" on page 89)
Table 86 Three plate/week auto target prep workflow—Day 5
Time Topic
8:00 Prepare Wash-Stain reagents for Plate B
8:30 Prepare GeneTitan reagent trays for Plate B (see "Stage 4: Preparation for the GeneTitan™ MC Instrument" on page 89)
9:30 Load GeneTitan reagent trays for Plate B
3:30 Prepare wash and stain reagents for Plate C
4:00 Prepare GeneTitan reagent trays for Plate C (see "Stage 4: Preparation for the GeneTitan™ MC Instrument" on page 89)
5:00 Load GeneTitan reagent trays for Plate C
280 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
8 Troubleshooting
Biomek FXP Target Prep Express
If a hardware problem is encountered while running the Axiom target preparation methods on the Biomek FXP Target Prep Express, you can do the following:
• See these documents:
– Biomek® Liquid Handler User’s Manual, Beckman Coulter Pub. No. 987834
– Biomek® Software User’s Manual v4.1, Beckman Coulter Pub. No. B30026AA
• For information on recovering a run, contact your Thermo Fisher Scientific Field Application Scientist.
• For additional information on Biomek FXP Target Prep Express hardware, error messages, or to request service, contact Beckman Coulter. Be sure to have the serial number of your workstation available.
GeneTitan™ Multi-Channel Instrument
See the GeneTitan™ Multi-Channel Instrument User Guide, Pub. No. 08-0306 for further troubleshooting information.
Table 87 GeneTitan Multi-Channel Instrument troubleshooting guidelines for the Axiom 2.0 Assay
Problem Possible causes Possible actions
Plate trapped in GeneTitan Multi-Channel Instrument.
• Plate (or plate with lid) not properly loaded in drawer.
• Notched edge of lid and plate not aligned.
• Gripper failed to retrieve plate.• System requires adjustment.
1. Restart the GeneTitan Multi-Channel Instrument.
2. Run the setup option Unload Plates.3. If the plate remains trapped in the
instrument, call Thermo Fisher Scientific support.
Computer frozen. • Too many processes running.• Attempting to transfer data while an
array plate is being scanned (imaged).
Restart the computer and unload all of the plates.• Plates in Hyb station: finish hybridization off-
line.• Plate in Scanner: rescan using Scan Only
function• Plate in Fluidics: use Wash/Scan Resume to
resume the fluidics process.Do not manually, or through the GCC transfer utility, move any data associated with the current plate that is being processed/scanned.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 281
Chapter 8 TroubleshootingGeneTitan™ Multi-Channel Instrument8
Miscellaneous messages
Hybridization aborted:• System-initiated abort• User-initiated abort
System-initiated abort:• Power lossUser-initiated abort:• User error• Other
Array plate and hybridization tray are still clamped:• Contact your local Field Service Engineer
with information on the workstation model.• The plate stack is moved to drawer 1.• Remove the plate stack and finish
hybridization offline.• Return the hybridized array plate stack to the
GeneTitan Multi-Channel Instrument and finish processing using the Wash/Scan process.
FAILED messages See "GeneTitan MC Instrument messages that appear when the instrument has a fluidics problem" on page 284
FLUIDIC DIAGNOSTIC messages
See "Fluidic diagnostic messages" on page 284.
Fluidics aborted:• System-initiated abort• User-initiated abort
System-initiated abort:• Power lossUser-initiated abort:• Incorrect protocol selected
Follow the recommendations and instructions under "Wash/Scan Resume" on page 288.
Table 87 GeneTitan Multi-Channel Instrument troubleshooting guidelines for the Axiom 2.0 Assay (Continued)
Problem Possible causes Possible actions
Table 88 Miscellaneous messages and recommended actions
Message and recommended action
Indicates that an item is in the gripper, and normal startup of the GeneTitan Multi-Channel Instrument is not possible. The item must be removed from the instrument before you can begin processing array plates.
Recommendation: click Yes.If you click No, nothing will occur. Homing will not complete and you will not be able to use the system.The item held by the gripper will be moved to either:• Drawer 2—plates and trays• Trash Bin—coversThe drawer names will reflect the location (left or right) and the drawer number (1 through 6).Examples: Drawer2L_Hta_DOWN = Scan tray on left side of drawer 2HtaHyb = Clamped hybridization tray and array plateDrawer(n)L/R_Hta_DOWN where n is the drawer number and L or R to indicate the left or right side.The _Hta_ (second term) indicates the item held. An example is drawer1R_HtaHyb_DOWN indicating it is an array plate with a hybridization tray or Drawer2L_ScanHta_Pk_DOWN indicating it is an array plate with a scan tray
282 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 8 TroubleshootingGeneTitan™ Multi-Channel Instrument 8
The drawer listed in the message is not fully closed. Manually push the drawer back into the instrument until it is fully closed. There are 2 stop positions with audible clicks; push until you hear the second click and the drawer is fully seated.
• Check that the array plate barcode has been entered correctly.
• Ensure that the library files required for the type of array plate you are using have been installed, and are installed in the correct directory.
• Restart the GeneTitan Instrument control software after library files have been installed.
Table 88 Miscellaneous messages and recommended actions (Continued)
Message and recommended action
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 283
Chapter 8 TroubleshootingGeneTitan™ Multi-Channel Instrument8
Fluidic diagnostic messages
Table 89 GeneTitan MC Instrument messages that appear when the instrument has a fluidics problem
Problem and possible causes
Rinse bottle—fluid level too low or bottle empty.
If this message is displayed:• during a water wash step, array processing has
been compromised.• during cleanup, array processing is okay, but
cleanup will not be complete.Always ensure that the GeneTitan bottles containing Wash A and Rinse are above the 50% mark when setting up the system to process an Axiom array plate.All 600 mL of the Wash Buffer B from the Axiom 2.0 reagent Kit should be emptied into the GeneTitan Wash B bottle when setting up the system to process a plate. This ensures that the GeneTitan Wash B bottle is filled to more than the requisite 35% of Wash B bottle volume.
About this message:• BUFFERX = Buffer bottle A, B or Rinse• WASHX = Wash A or B reservoir in the fluidics
station.Recommended actions:• Replenish fluid level in the Rinse or Wash Bottle B
to the 1L mark. Do not overfill.– Only replenish bottles when prompted by the UI.
Replenishing during fluidic processing may cause system malfunction including overflowing inside the system and more problems. The only thing to do while a plate is running is to ensure that bottle caps are secure.
• Replenish fluid level in Wash Bottle A to 2L.• Secure the bottle cap.• Replace the filterInstructions for filter replacement in the GeneTitan™ Multi-Channel Instrument User Guide, Pub. No. 08-0308.If the problem persists, call Thermo Fisher Scientific Support.
284 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 8 TroubleshootingGeneTitan™ Multi-Channel Instrument 8
The typical cause is an unsecure bottle cap.
If the failure is detected during priming, the instrument will pause and wait for the problem to be corrected.
If the failure is detected during another process, and if the cause is a clogged filter, wait until the end of the run to replace the filter.Instructions for filter replacement in the GeneTitan™ Multi-Channel Instrument User Guide, Pub. No. 08-0306.
When the instrument experiences a loss in Clean Dry Air (CDA) pressure, the software will display the warning message.
When the pressure is detected again, a dialog message confirming the availability of CDA pressure is displayed.
Possible causesVerify that the facility CDA or the portable CDA compressor is in working condition. See the GeneTitan MC Instrument Site Preparation Guide for the portable compressor model that has been validated with the GeneTitan MC instrument.Contact your local Field Service Engineer and notify them about the error message.
Table 89 GeneTitan MC Instrument messages that appear when the instrument has a fluidics problem
Problem and possible causes
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 285
Chapter 8 TroubleshootingGeneTitan™ Multi-Channel Instrument8
Leak DetectedLeak checks are performed at application startup and any time a fluidic process (priming filling draining etc.) is performed. The leak detection is a hard-wired sensor which will shut off fluid flow without software control. Leaks are normally confined to the drip pan located inside the system.
Causes:• System malfunction• User killing the application using task manager
during a fill operation resulting in application exit without stopping flow.
Solution: Contact Support. The system cannot be used for any fluidic processing until this is resolved.
Leak Resolved This message is displayed when the leak is resolved (meaning the sensor LED is again lit up). If the original leak detected message was not acknowledged it will be automatically removed from the GUI and replaced by the following message. It will remain displayed until another leak is detected or the user acknowledges it by pressing OK. To resolve this issue complete the following tasks• Verify all internal and external tubing is connected
and clean• Verify wash reservoirs are clean• Verify all bottle caps are secure and that no bottle
cap is crimping a supply line.• Verify vacuum is working properly• Do not refill bottles or empty waste except when
prompted to by the GeneTitan application.• Contact your facility group to ensure CDA is
supplied to your GeneTitan system.Contact your Thermo Fisher Scientific Field Service Engineer to have the sensor adjusted or replaced if the problem persists even after correcting for the usual causes outlined above.
Table 89 GeneTitan MC Instrument messages that appear when the instrument has a fluidics problem
Problem and possible causes
286 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Chapter 8 TroubleshootingGeneTitan™ Multi-Channel Instrument 8
Filter Error Message: Dispense related check
Filter Error Message: Fill related check
The filters in the GeneTitan fluidics bottles (Wash A, Wash B and Rinse) need to be replaced when the filters are worn out. The software displays warning message boxes for the filter in each reagent bottle when it detects a problem or shows a trend of increased fill times during fluid fill operations. If an error is detected as described above, then a message box titled “Filter Change Required” is displayed along with the information on the specific dispense operation. You should change all 3 filters when a warning is displayed for any 1 of the 3 filters.See the section "Replacing the filter" on page 314 in Appendix F.
Table 89 GeneTitan MC Instrument messages that appear when the instrument has a fluidics problem
Problem and possible causes
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 287
Chapter 8 TroubleshootingGeneTitan™ Multi-Channel Instrument8
Wash/Scan Resume
If a run is aborted during fluidics processing, the instrument will place the aborted array plate into the scan tray. To restart this process, remove the Axiom array plate from the scan tray and place the array in its protective blue base.
The step at which the run was aborted can be identified by:
• Viewing the System Status window if you are aborting the last plate through the fluidics system.
• Initiating the resume process.
1. System Setup tab: Select Wash/Scan Resume
2. Follow the prompts to unload and reload all drawers.
The trays will be loaded. It is up to you to determine whether or not to load fresh reagents or reuse the trays already in the GeneTitan Multi-Channel Instrument. Base your decision upon the step where the problem occurred.
To help ensure that the samples are processed correctly, we recommend that you:
1. Load new stain trays with fresh reagents.
2. Load a new scan tray.
We do not recommend the use of trays without reagents or holding buffers for steps that appear to have already executed.
Resume stepYou must select the step at which you wish to resume plate processing. You can select any step that has not yet been started.
For certain steps, you can enter a duration in seconds (even if the step requires >1 hour to run, you must enter the duration in seconds). You can set a step for less time than normal, but not for longer than the normal duration.
Aborting a run • Abort can take up to 3 minutes if a plate is in the fluidics station. Status window Abort Requested changes to Abort Completed.
• Clamped Array-Plate-Hybridization tray stack that is aborted from the oven or from drawerIN (drawer 6) is moved to drawer 1.
• Proceed as follows:
– Use the Unload Plates option to remove the aborted plate(s).
– Start another run which will force an unload of the aborted plate(s)
System-initiated• Power interruption
• Plate loaded incorrectly
• Equipment malfunction
The system will abort the processing. Follow the instructions displayed in the user interface.
User-initiatedCan abort processing of individual array plates.
If multiple plates are being processed, the gripper may continue to process the remaining array plates.
288 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
A Safety
General safety
For research use only. Not recommended or intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals.
WARNING! GENERAL SAFETY. Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device. Ensure that anyone using this product has received instructions in general safety practices for laboratories and the safety information provided in this document.
• Before using an instrument or device, read and understand the safety information provided in the user documentation provided by the manufacturer of the instrument or device.
• Before handling chemicals, read and understand all applicable Safety Data Sheets (SDSs) and use appropriate personal protective equipment (gloves, gowns, eye protection, etc). To obtain SDSs, see the ʺDocumentation and supportʺ section in this document.
WARNING! The following components contain harmful or toxic ingredients:
• Axiom Stabilize Soln: 8% Gluteraldehyde
• Axiom HybSoln 2: 100% Formamide
• Axiom Hyb Buffer: <55% Tetramethylammonium Chloride
In all cases customers should use adequate local and general ventilation in order to minimize airborne concentrations.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 289
Appendix A SafetyChemical safetyA
Chemical safety
WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensure laboratory personnel read and practice the general safety guidelines for chemical usage, storage, and waste provided below, and consult the relevant SDS for specific precautions and instructions:
• Read and understand the Safety Data Sheets (SDSs) provided by the chemical manufacturer before you store, handle, or work with any chemicals or hazardous materials. To obtain SDSs, see the ʺDocumentation and supportʺ section in this document.
• Minimize contact with chemicals. Wear appropriate personal protective equipment when handling chemicals (for example, safety glasses, gloves, or protective clothing).
• Minimize the inhalation of chemicals. Do not leave chemical containers open. Use only with adequate ventilation (for example, fume hood).
• Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the manufacturerʹs cleanup procedures as recommended in the SDS.
• Handle chemical wastes in a fume hood.
• Ensure use of primary and secondary waste containers. (A primary waste container holds the immediate waste. A secondary container contains spills or leaks from the primary container. Both containers must be compatible with the waste material and meet federal, state, and local requirements for container storage.)
• After emptying a waste container, seal it with the cap provided.
• Characterize (by analysis if necessary) the waste generated by the particular applications, reagents, and substrates used in your laboratory.
• Ensure that the waste is stored, transferred, transported, and disposed of according to all local, state/provincial, and/or national regulations.
• IMPORTANT! Radioactive or biohazardous materials may require special handling, and disposal limitations may apply.
290 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Appendix A SafetyBiological hazard safety A
Biological hazard safety
WARNING! BIOHAZARD. Biological samples such as tissues, body fluids, infectious agents, and blood of humans and other animals have the potential to transmit infectious diseases. All work should be conducted in properly equipped facilities using the appropriate safety equipment (for example, physical containment devices). Safety equipment also may include items for personal protection, such as gloves, coats, gowns, shoe covers, boots, respirators, face shields, safety glasses, or goggles. Individuals should be trained according to applicable regulatory and company/ institution requirements before working with potentially biohazardous materials. Follow all applicable local, state/provincial, and/or national regulations. The following references provide general guidelines when handling biological samples in laboratory environment.
• U.S. Department of Health and Human Services, Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th Edition, HHS Publication No. (CDC) 21-1112, Revised December 2009; found at:www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf
• World Health Organization, Laboratory Biosafety Manual, 3rd Edition, WHO/CDS/CSR/LYO/2004.11; found at:www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 291
B Fragmentation quality control gelprotocol
Protocol for running a fragmentation quality control gel
Equipment required
E-Gels and reagents
Consumables
Table 90 Equipment required
Item Supplier Part number
Gel imager Your choice —
Pipette, multichannel or single channel P20 Your choice —
Plate centrifuge Your choice —
Vortex Your choice —
Table 91 E-Gel and reagents required
Item Supplier Part number
Mother E-Base Device
Life Technologies(formerly
Invitrogen)
EB-M03
Daughter E-Base Device EB-D03
E-Gel® 48 4% agarose gels G8008-04
TrackIt 25 bp DNA Ladder 10488-022
TrackIt Cyan/Orange Loading Buffer 10482-028
Nuclease-free Water Your choice —
Table 92 Gel and reagents required
Item Supplier Part number
Adhesive film – use 1 of the following:• MicroAmp Clear Adhesive Film• Microseal 'B' Film
Thermo Fisher ScientificBio-Rad
4306311MSB1001
Pipette Tips Same brand as pipette —
292 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Appendix B Fragmentation quality control gel protocolProtocol for running a fragmentation quality control gel B
Diluting the TrackIt™ Cyan/Orange Loading Buffer
The following recipe is for preparing a 1000-fold dilution of the TrackIt Cyan-Orange Loading Buffer.
To dilute the TrackIt Cyan/Orange Loading Buffer:
1. Add 50 µL of TrackIt Cyan/Orange Loading Buffer to 49.95 mL nuclease-free water.
Total volume 50 mL.
2. Vortex tube to mix well.
3. Store at room temperature.
To dilute the TrackIt 25bp Ladder:
The following recipe is for preparing a 15-fold dilution of the Invitrogen TrackIt 25 bp DNA Ladder.
1. In a 1.5 mL microcentrifuge tube, add 6 µL of TrackIt 25 bp Ladder to 84 µL nuclease-free water. Total volume: 90 µL.
2. Vortex tube to mix well. Centrifuge to get droplets down.
Note: The recipe has enough volume to fill 4 marker wells of 1 E-Gel® 48 4% agarose gel. Scale up as needed if running multiple gels.
Fragmentation QC gel protocol
This protocol is based on running QC gels for 96 samples.
To run a fragmentation QC gel:
1. Tightly seal the gel QC plate prepared during automated target preparation.
2. Vortex the center of the plate for 3 sec. Centrifuge to 1,000 rpm to get droplets down.
3. Connect an E-Base™ device(s) to an electrical outlet.
4. Push the Power/Prg button on each to ensure the program is in EG mode (not EP mode).
5. Take the gel out of the pouch and remove the combs.
6. Place the E-Gel® 48 gel into an E-Base unit.
7. Load 20 µL from each well of the Gel QC plate onto the gels.
8. Load 15 µL of 15-fold diluted TrackIt 25 bp ladder into the marker wells (M).
9. Load 20 µL nuclease-free water into any unused wells.
10. Run the gels for 22 minutes.
11. Image the gel.
Fragmentation QC gel images should look similar to the gel shown in Figure 153.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 293
Appendix B Fragmentation quality control gel protocolProtocol for running a fragmentation quality control gelB
Figure 153 Example of a typical fragmentation QC E-gel
125 bp
25 bp
25 bp
125 bp
Fragments should fall between 125 bp and 25 bp.
25 bp ladder 25 bp ladder
294 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
C Sample quantitation afterresuspension
Protocol for sample quantitation after resuspension
Equipment required The following equipment is required for this protocol.
Quantitate the diluted samples
During target prep, 2 plates of diluted samples are prepared: 1 for OD quantitation and 1 for a QC gel to check the fragmentation reaction.
For OD quantitation, readings should be taken at wavelengths of 260, 280, and 320 nm. See ʺSuggested protocol for OD quantitation using the DTX 880ʺ on page 297 for more information.
To quantitate the diluted samples prepared for OD quantitation:
1. Launch the Multimode Analysis Software.
2. When the Protocol Selection List is displayed, select the appropriate protocol.
3. Right-click the protocol and select Run the selected protocol.
4. In the Result Name field, enter your experiment name.
5. Click the Eject Plate Carrier icon.
6. Load the OD plate onto the DTX 880.
7. Click the Close Plate Carrier icon.
8. Click the Run the Selected Protocol icon at the bottom of the window.
When the protocol is finished running, a list of results is displayed. If you used the formula provided in this appendix, 2 XML files are generated (Figure 154). Open the ResultData file with Microsoft® Excel® to view and assess the OD readings. RawData file information is included in the ResultData file.
Table 93 Equipment required for sample quantitation after resuspension
Quantity Item
1 DTX 880 Multimode Detector with genomic filter slide
Figure 154 List of files that are generated post DTX-880 scan.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 295
Appendix C Sample quantitation after resuspensionProtocol for sample quantitation after resuspensionC
Assess the OD readings
If using the formula provided in this appendix, the raw data is included in the final Result Data file. Figure 155 is an example of a Result Data file. Your OD readings should be similar to those displayed below.
OD yield assessment guidelinesThe measurement of the yield of DNA after resuspension of the pellets is an important QC checkpoint in the Axiom 2.0 Assay. If the median yield for the plate is < 1200 µg DNA per sample:
• Pause the protocol.
• Assess each of the steps performed to that point to determine the possible source of the low yields.
This DNA yield corresponds to an A260 value of approximately 0.59 and an A260-A320 value of approximately 0.50.
Figure 155 Example of Result data file with acceptable OD readings
296 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Appendix C Sample quantitation after resuspensionSuggested protocol for OD quantitation using the DTX 880 C
Suggested protocol for OD quantitation using the DTX 880
The formula suggested below requires 6 passes. The settings and formula are shown below.
Protocol Type—Analysis
General Settings—Enter a name for the protocol
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 297
Appendix C Sample quantitation after resuspensionSuggested protocol for OD quantitation using the DTX 880C
Technique Type—Select Absorbance
Labware—x_Abs_Greiner 96 UV clear std (96 Microplate Format)
298 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Appendix C Sample quantitation after resuspensionSuggested protocol for OD quantitation using the DTX 880 C
Layout Settings—as appropriate for 96-array format plates
Method Selection—add (+) the 3 formulas created on the Data Reduction Page to the Group 1 box.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 299
Appendix C Sample quantitation after resuspensionSuggested protocol for OD quantitation using the DTX 880C
Data Reduction Page—create the formulas required for scans at 260, 280 and 320This protocol consists of 6 passes. Click Add new Pass to create passes 2 through 6, shown in these figures below.
300 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Appendix C Sample quantitation after resuspensionSuggested protocol for OD quantitation using the DTX 880 C
1
k
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 301
Appendix C Sample quantitation after resuspensionSuggested protocol for OD quantitation using the DTX 880C
302 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Appendix C Sample quantitation after resuspensionIf performing sample quantitation on a plate reader other than the DTX880 C
Output Settings—select Export to Microsoft® Excel® and Show Result Viewer
Save the protocol.
If performing sample quantitation on a plate reader other than the DTX880
Your plate reader should be calibrated to ensure accurate readings.
The total yield in µg per well can be calculated as:
(A - C)*D*V*E/P
Where:
A = the observed OD260
C = the observed OD320 (an estimate of a blank reading)
D = 120 (the net dilution factor when preparing the OD Sample plate as described in the Automated Target Preparation Protocol)
V = 115 (the volume of the sample in µL after the resuspension step)
E = 0.05 (the extinction coefficient of duplex DNA at 260 nm)
P = the optical path length for the plate type and plate reader used.
If your plate reader does not record the OD320, the OD260 of a blank solution of water only should be used for the parameter “C” above.
The optical path length is dependent on the type of plate and spectrophotometer used. Check your manufacturerʹs recommendations for the path length for your instrument and plate type or for recommendations on how to measure this quantity. The SpectraMax Plus384, described as an alternative spectrophotometer in the Assay 96-Array Format Automated Workflow for Beckman Biomek FXP Site Preparation Guide, Pub. No. 702984, can employ an automated path length detection system. Consult this instrument’s manual for more information.
The resulting yield calculations can be compared against the typical yields shown in column H of Figure 155 on page 296 and against ʺOD yield assessment guidelinesʺ on page 296.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 303
D Registering samples in GeneChip™
Command Console™
Creating a GeneTitan™ Array Plate Registration file
A GeneTitan Array Plate Registration file is a Microsoft® Excel® spreadsheet that includes information on the samples you are processing on a single array plate. This information includes the array plate format, the array plate barcode, and sample file names so that you can track the samples that are loaded onto a particular array plate.
The version of Microsoft Excel must be 1997-2000 (file extension is .xls; not .xlsx).
To create a GeneTitan Array Plate Registration file:
1. In GCC Portal, open the Samples menu and select GeneTitan Array Plate Registration (Figure 156).
Figure 156 Selecting GeneTitan Array Plate Registration
304 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Appendix D Registering samples in GeneChip™ Command Console™
Creating a GeneTitan™ Array Plate Registration file D
2. Select the array plate to be processed on the GeneTitan MC Instrument (Figure 157 on page 305).
a. Select the array plate type.
b. Click Download.
3. Complete the registration file as follows:
a. Click the Microsoft Excel box on the bottom bar of the monitor to open the Excel spreadsheet.
b. Enter a unique name for each sample (Sample File Name) and any additional information you would like to include (Figure 158).
c. Do one of the following:
• If you are ready to load the array plate onto the GeneTitan MC Instrument, scan the array plate barcode and proceed to the next step.
• If you are not ready to load the array plate onto the GeneTitan MC Instrument, proceed directly to the next step.
4. Save the file as follows:
a. Open File Save As.
b. Enter a name for the array plate registration file.
c. Click Save.
By default, the file is saved in the Affymetrix_Downloads folder.
Figure 157 Selecting the type of array plate to be processed
Figure 158 Entering sample information into an Array Plate Registration file
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 305
Appendix D Registering samples in GeneChip™ Command Console™
Creating a GeneTitan™ Array Plate Registration fileD
5. When ready to load the array plate onto the GeneTitan MC Instrument:
a. Click the Browse button, navigate to the file, and click Open.
b. Scan the array plate barcode if not already scanned.
c. Click the Upload button (Figure 159), wait for the information to load, then click the Save button located at the bottom of the next page that is displayed.
If the samples are successfully registered, the message in Figure 160 is displayed.
Figure 159 Uploading the Array Plate Registration file to GCC
Figure 160 Array plate samples successfully registered
306 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
E Deionization procedure forGeneTitan™ trays and covers
We recommend the use of the Zerostat 3 Anti-Static Gun (Cat. No. 74-0014) to deionize GeneTitan™ MC Instrument stain tray trays and lids.
Deionize the inner surface of each tray and cover:
• The surface of the tray with the wells that will hold reagents.
• The surface of the cover that will face the reagents.
IMPORTANT! Except for the Axiom™ array plates, scan tray and the hybridization tray, you must deionize all GeneTitan stain trays, stain tray covers and scan tray cover using an anti-static gun. You must do this before you fill the trays with reagents and before you place the covers on the trays. Deionization removes the static electricity. The presence of static electricity on the underside of the cover can cause the gripper to lift the tray along with the tray cover and can result in an aborted run. See Figure 161, Figure 162 and Figure 163.
CAUTION! Do not deionize the scan tray or hybridization tray.
Figure 161 Scan tray with cover. Deionize only the cover.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 307
Appendix E Deionization procedure for GeneTitan™ trays and coversDeionization procedureE
Deionization procedure
The following process provides guidance on how to use the anti-static gun on the stain and scan tray covers only. See Figure 163.
1. Treat the plate or lid as if it were divided into 6 sections (see Figure 163), and deionize as follows.
2. Place a Kimwipes laboratory tissue on the benchtop.
3. Place the stain tray on a table top. Use the anti-static gun to aim at the center of each of the 6 sections on a 96-well tray and pull the trigger. Ensure that a stream of ionized particles settles on all wells of the stain tray to dissipate the static electricity. Squeeze and release the trigger slowly 3 times over each section (Squeeze for approximately 2 seconds and release for approximately 2 seconds).
4. Place the stain tray cover with the flat surface facing upward on the Kimwipes laboratory tissue.
5. Aim the anti-static gun (Cat. No. 74-0014) approximately one-half inch away from the flat surface and pull the trigger. As you pull the trigger move the gun across the cover so that the stream of ionized particles settles on all areas of the cover and dissipates the static electricity. Squeeze and release the trigger slowly 3 times over each section (squeeze for approximately 2 seconds and release for approximately 2 seconds).
Figure 162 Stain tray with cover. Deionize the cover and the tray.
WARNING! The deionization steps 4 and 5 will damage the Axiom arrays on the plate. Before using the anti-static gun, ensure that the Axiom array plates remain in their protective pouch and placed away from the deionization area. You must place the scan tray and hybridization tray away from the area where you are performing deionization.
308 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Appendix E Deionization procedure for GeneTitan™ trays and coversDeionization procedure E
6. Place the treated cover or tray on the tissue and lift it up (see Figure 163).
7. Do one of the following:
• If the tissue does not cling to the plastic, proceed with the protocol.
• If the tissue still clings to the plastic, then perform steps 3 and 4 again. If it continues to cling to the plastic, test the device using the ion-indicator cap to confirm that the unit is still releasing ions. Otherwise, it may be time to replace the unit.
Figure 163 Removing the static charge from stain trays and lids.
Treat the inside surface of stain trays (right) and cover (left).
• If a tissue clings to treated surface, try the deionization procedure again. • If the tissue still clings, it may be time to replace the anti-static gun.
1 2
3 4
5 6
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 309
Appendix E Deionization procedure for GeneTitan™ trays and coversIon-indicator capE
Ion-indicator cap
The ion-indicator cap is a testing device used to verify the release of ions when the anti-static gun is in use (Cat. No. 74-0014, Figure 163).
Testing the Zerostat 3 with the ion-indicator cap
1. Insert the ion-indicator cap into the nose of the Zerostat and then slowly squeeze the release trigger (see Figure 164).
2. Observe the discharge through the viewing slot on the ion-indicator cap of the anti-static gun. A visible light is observed in the viewing window on the cap when charged ions are discharged.
3. If you cannot see the light, the gun may be unusable and you should replace it.
4. Each Zerostat anti-static gun is capable of 50,000 trigger operations, which is sufficient for approximately 200-250 runs on the GeneTitan MC Instrument.
Figure 164 Zerostat 3 anti-static gun (Cat. No. 74-0014) with ion-indicator cap to test functionality
IMPORTANT! Ensure to remove the cap from the gun before deionizing a tray or cover.
Ion-indicator cap
The Ion-Indicator Cap is attached to the Zerostat to test the functionality of the anti-static gun.
IMPORTANT: Do not leave the Ion-Indicator Cap on the Zerostat gun when deionizing trays and lids.
310 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
F GeneTitan™ Multi-ChannelInstrument Care
Cleaning and maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
Servicing the outer enclosure fan filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
Replacing the bottle filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313
Replacing the xenon lamp in the GeneTitan™ MC Instrument . . . . . . . . . . . . . 315
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320
This chapter provides instructions on caring for and maintaining the instrument and on troubleshooting if problems arise.
• Always run a Shutdown protocol when the instrument will be off or unused overnight or longer. This will prevent salt crystals from forming within the Fluidics system.
• Always use deionized water to prevent contamination of the lines. Swap out old buffers with freshly prepared buffer at each system startup.
The GeneTitan™ Instrument should be positioned on a sturdy level bench away from extremes in temperature and away from moving air.
Cleaning and maintenance
The GeneTitan family of instruments require little in the way of customer maintenance. The instruments must be kept clean and free of dust. Dust buildup can degrade performance. Wipe the exterior surfaces clean using a mild dish detergent solution in water. Do not use ammonia based cleaners or organic solvents such as alcohol or acetone to clean the system because they may damage the exterior surfaces.
The following tasks should be performed regularly to ensure the Imaging Device remains in working order.
Monthly Wipe down the outer surface of the Imaging Device with a dry cloth.
Every 6 months Replace the cooling fan air filters at the rear of the instrument.
Replace the Micropore filters in the Wash A, Wash B, and Rinse bottles. If you run 4-8 plates/week then the micro-pore filters need to be replaced more frequently.
IMPORTANT! Before performing maintenance turn off power to the instrument to avoid injury in case of an electrical malfunction.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 311
Appendix F GeneTitan™ Multi-Channel Instrument CareServicing the outer enclosure fan filtersF
Servicing the outer enclosure fan filters
Cleaning schedule The GeneTitan fan filter cartridge (Figure 165) should be cleaned at least every 90 days of service. Note that in some service locations, the presence of excessive dust or particulate matter may necessitate cleaning the cartridge more often than 90 days.
A plugged filter cartridge can cause excessive temperatures within the machine that can cause unwanted evaporation of GeneTitan reagents.
Part details for GeneTitan fan filter:Thermo Fisher Scientific Cat. No. 01-0669
Number of filters required per GeneTitan instrument: 3
Cleaning procedure 1. Slide the filter cartridge from the fan filter cartridge at the rear of the GeneTitan MC Instrument.
2. Submerse in clean DI water. Rinse and agitate gently to dislodge material.
3. Remove from water and dry with clean compressed air or towels.
4. When the filter cartridge is completely dry to the touch, re-install the cartridge.
Figure 165 The GeneTitan filter cartridge
312 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Appendix F GeneTitan™ Multi-Channel Instrument CareReplacing the bottle filters F
Replacing the bottle filters
The bottles used in GeneTitan MC Instrument contain a filter to remove particulates that may exist in the buffers and DI water. The filters in the GeneTitan fluidics bottles (Wash A, Wash B and Rinse) need to be replaced when the filters are clogged.
The message boxes displayed in Figure 166 will provide information on fluid dispense errors that were detected by the instrument for any of the bottles or when the instrument detects an increase in the amount of time that is required to perform the fill operations.
If an error is detected as described above, then a message box titled “Filter Change Required” is displayed (Figure 166) along with the information on the specific dispense operation. You should change all 3 filters when a warning is displayed for any 1 of the 3 filters.
Note: The reagent bottles are depressurized when this warning message is displayed. It is safe to change the filters in all 3 fluidic bottles when this message is displayed.
After changing the filters in all 3 bottles using the procedure described below, press the Yes button to continue. If you choose to ignore the error message, press the No button. This warning message will be displayed each time GCC instrument control software is launched. You may also experience data quality issues if particulate matter cannot be trapped by the filters because they are clogged.
Figure 166 Filter Change Required messages
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 313
Appendix F GeneTitan™ Multi-Channel Instrument CareReplacing the bottle filtersF
We recommend that your site keep 3 spare filters on hand in the event the filters need to be replaced. The procedure for replacing the filters is simple.
GeneTitan reagent bottle filters part details:Thermo Fisher Scientific Cat. No. 01-0671
Removing and inspecting the filter
1. Loosen and remove the cap on the bottle.
2. Carefully remove the filter from the end of the filter body.
3. Visually inspect the filter. If one of the filters appears to have a concentration of dirt or contaminate in it, discard it and replace the filter with a new one.
Replacing the filter 1. Insert the filter into the end of the filter body.
2. Replace the cap onto the bottle and tighten it.
3. Repeat for each bottle.
Figure 167 Replacing the filter
Buffer supply line
Filter holder
Filter
1
2
3
1 2 3
IMPORTANT! Replace one filter at a time to ensure the correct connection of the buffer supply tube to its respective bottle. The color of the buffer supply tubing matches the bottle color code.
314 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Appendix F GeneTitan™ Multi-Channel Instrument CareReplacing the xenon lamp in the GeneTitan™ MC Instrument F
Replacing the xenon lamp in the GeneTitan™ MC Instrument
This section applies to your site only if you have the GeneTitan Multi-Channel (MC) instrument. After the normal life expectancy of the lamp has expired, the software application will alert you to the requirement to replace the lamp. This procedure is simple but you must follow good health and safety precautions.
GeneTitan xenon lamp catalog number: Thermo Fisher Scientific 01-0740
Lamp life/imaging device status notices
The Imaging Status pane displays lamp life and Imaging Device status notices for the GeneTitan MC Instrument.
In normal operation, the pane displays the hours of life left in the lamp (Figure 168):
It displays a red or yellow notice when the lamp life is getting short (Figure 169):
It also displays a red notice when the Imaging Device is offline (Figure 170):
Note: The 300 watt xenon lamp in the GeneTitan MC Instrument is warranted for 500 hours. The instructions to replace the lamp are available on the following page. After changing the lamp, it is necessary to reset the lamp life clock manually.
IMPORTANT! Do not try to replace the lamp when a plate is being processed either in the fluidics or scanner system.
Figure 168 Lamp Life above tolerance
Figure 169 Lamp Life above tolerance
Figure 170 Imaging Device offline
WARNING! You must turn off the lamp using the power switch in the rear of the unit and remove the power cord. Allow the lamp to cool before attempting to replace the lamp.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 315
Appendix F GeneTitan™ Multi-Channel Instrument CareReplacing the xenon lamp in the GeneTitan™ MC InstrumentF
Removing the xenon lamp
1. Unscrew the 4 retaining bolts. They should be finger tight (Figure 171).
2. Remove and set aside the warning cover to reveal the xeon lamp contained within.
3. Place each hand on each side of the blue plastic flange and lift out the lamp in a vertical motion (Figure 172). You must use both hands to remove the lamp successfully. Apply equal pressure on each side of the lamp and gently lift.
Figure 171 Unscrewing the Bolts
Figure 172 Lifting out the lamp
Unscrew these 4 bolts.
316 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Appendix F GeneTitan™ Multi-Channel Instrument CareReplacing the xenon lamp in the GeneTitan™ MC Instrument F
Replacing the lamp
1. Hold the lamp by the blue plastic flanges. Ensure that the lamp bulb faces inward toward the reflecting mirror (Figure 173) and vertically insert the lamp (Figure 174).
2. Replace the warning cover and hand tighten the bolts (Figure 171).
CAUTION! Ensure that you install the lamp in the correct orientation.
Figure 173 The reflecting mirror
Reflecting mirror
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 317
Appendix F GeneTitan™ Multi-Channel Instrument CareReplacing the xenon lamp in the GeneTitan™ MC InstrumentF
Figure 174 Inserting the lamp
IMPORTANT: The lamp bulb faces away from the fan and toward the reflecting mirror.
318 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Appendix F GeneTitan™ Multi-Channel Instrument CareReplacing the xenon lamp in the GeneTitan™ MC Instrument F
Resetting the lamp counter
You must alert the software application that you have replaced the lamp so that the hours of the lamp counter are reset to zero. This menu option is only available when the system is not processing any plates.
1. On the software application click Tools Reset Counter for Life Remaining (Figure 175).
2. The software will display a message that asks you to confirm the lamp life counter is being reset as a result of lamp replacement (Figure 176).
Figure 175 Inserting the lamp
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 319
Appendix F GeneTitan™ Multi-Channel Instrument CareTroubleshootingF
3. Click Yes if you want to reset the counter. The software will display a message that confirms that the software has reset the counter (Figure 177).
Troubleshooting
This section provides instructions on how to identify and solve simple problems with the GeneTitan MC Instrument. If a problem or error occurs that is not listed in this chapter contact Thermo Fisher Scientific Technical Support for assistance.
For software errors that do not involve hardware crashes the most common solution is to shut down the application and then restart it. If the same error occurs shut down both the application and the computer and then restart. If it still occurs shut down the GeneTitan MC Instrument and then restart.
Log files The log files are produced by different GCC components. The logs provide a record of the tasks performed by different components, such as the migration tools and installer. These log files provide useful information for troubleshooting problems. These files may be requested by your Field Application Scientist (FAS), Field Service Engineer (FSE), or the call center.
Figure 176 Are you sure?
Figure 177 The counter is reset.
320 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Appendix F GeneTitan™ Multi-Channel Instrument CareTroubleshooting F
GCC log filesThe following files are generated by the GeneTitan Instruments. All the GCC log files are from the following path: C:\Command_Console\Logs. The different log files include:
Other GCC filesYour FAS and/or FSE may request you to send the following files for troubleshooting:
1. Library files (*.PARAMS, *.MASTER, *.WORKFLOW, *.SMD, *.MEDIA) located in C:\Command_Console\Library, excluding the large analysis library files (CDF, PSI, GRC).
2. Provide a list of all sub folders and their contents under the library files folder located in C:\Command_Console\Library. Ensure there are no duplicate library files, as these can cause problems.
3. GCC system configuration file located at C:\Command_Console\Configuration\Calvin.System.config
4. Pending job order files located in C:\Command_Console\Jobs
5. Other GCC related information, such as:
a. The number of files under C:\Command_Console\Data, including sub directory.
b. If the system is a networked system or a standalone system.
c. Other applications installed on the system, such as antivirus application, MS Office, and Internet Explorer versions.
GCC log files for GeneTitan™ MC Instrument Systems
Log files for the GeneTitan MC Instrument control processes are placed in subdirectories of the C:\Command_Console\Logs\ folder. Thermo Fisher Scientific may need the following files for troubleshooting:
GeneTitan MC Instrument fluidics
1. C:\Command_Console\Logs\96F\
a. Subdirectories named by date (e.g., Log7-29-2009)
• Collect all dated directories and contents since the GeneTitan application was started, not just the date of the event (some logging goes into files from the date the application started so this can be critical for us).
• Absolutely required are all the log directories from the date the run was started to the date of the event.
2. C:\Command_Console\Logs\96F\FluidicErrorLog - all files in this directory.
Systemlog.XML XML file with system information.
DEC.log Text file with information on the use of the Data Exchange Console (DEC).
DECError.log Text file with information on errors created while using DEC.
AGCC_LibFileImporter. log (with date and time code)
Text file with info on use of the Library File Importer.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 321
Appendix F GeneTitan™ Multi-Channel Instrument CareTroubleshootingF
GeneTitan MC Instrument imaging device
1. C:\Affymetrix\GeneChipHTScanControlMC\Log - collect all dated directories and contents since the GeneTitan application was started.
2. C:\Affymetrix\GeneChipHTScanControlMC\RunLog - collect all dated directories and contents since the GeneTitan application was started.
Insufficient disk space notice
If there is not enough memory on the computer’s drives to save the data from an array plate, a notice appears (Figure 178) when:
• you first initialize the software and instrument.
• you select arrays for imaging.
If you see this notice, you will need to free up sufficient disk space before imaging starts.
Figure 178 Insufficient disk space notice
322 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Documentation and support
Related documentation
Table 94 Documents related to the Axiom™ 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP
Document Publication number Description
Axiom™ 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP Site Preparation Guide
702984 Provides guidance on reagents, instruments, and supplies required to run the Axiom 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP (Windows XP) QRC
702962 An abbreviated reference for the target preparation step of the Axiom 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP running on the Windows XP operating system. This quick reference document is intended for experienced users.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP (Windows® 7) QRC
703369 An abbreviated reference for the target preparation step of the Axiom 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP running on the Windows 7 operating system. This quick reference document is intended for experienced users.
Axiom™ 2.0 gDNA Sample Prep Protocol QR
702987 An abbreviated reference on the genomic DNA sample preparation protocol.
Axiom™ gDNA Sample Prep for Genome-Wide BOS 1 Array Plate QR
702975 An abbreviated reference on the genomic DNA sample preparation protocol for the Genome-Wide BOS 1 Array Plate.
GeneTitan™ MC Protocol for Axiom™ 2.0 Array Plate Processing QR
702988 An abbreviated reference for processing Axiom 2.0 array plates with the GeneTitan Multi-Channel Instrument.
GeneTitan™ Multi-Channel Instrument User Guide
08-0308 The GeneTitan Multi-Channel (MC) Instrument automates array processing from target hybridization to data generation by combining a hybridization oven, fluidics processing, and state-of-the art imaging device into a single bench-top instrument. This document detailing the use, care, and maintenance for the GeneTitan MC Instrument.
GeneTitan™ Multi-Channel Instrument Site Preparation Guide
08-0305 Provides guidance on creating and maintaining the proper environment required for the GeneTitan Multi-Channel Instrument.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 323
Documentation and supportRelated documentation
Analysis and Software
Axiom™ Genotyping Solution Data Analysis Guide
702961 This guide provides information and instructions for analyzing Axiom genotyping array data. It includes the use of Axiom™ Analysis Suite, Applied Biosystems Microarray Power Tools (formerly APT) and SNPolisher R package to perform quality control analysis (QC) for samples and plates, SNP filtering prior to downstream analysis, and advanced genotyping methods.
Applied Biosystems ™ GeneChip™ Command Console™ Software User Guide
702569 This user guide provides instructions on using Applied Biosystems GeneChip Command Console Software (GCC) used to control GeneChip instrument systems. Command Console Software provides an intuitive set of tools for instrument control and data management used in the processing of GeneChip Arrays.
Axiom™ Analysis Suite User Guide 703307 This user guide provides instructions on using Axiom™ Analysis Suite—a single-source software package to enable complete genotyping analysis of all Axiom arrays.
Axiom 2.0 Assay Manual Protocol
Axiom™ 2.0 Assay 96-Array Format Manual Protocol User Guide
702990 This user guide provides comprehensive instructions on running the Axiom 2.0 Assay 96-Array Format Manual Protocol.
Axiom™ 2.0 Assay 96-Array Format Manual Protocol Site Preparation Guide
702991 Provides guidance on reagents, instruments, and supplies required to run the Axiom 2.0 Assay 96-Array Format Manual Protocol.
Axiom™ 2.0 Assay 96-Array Format Manual Protocol QRC
702989 An abbreviated reference for the target preparation step of the Axiom 2.0 Assay 96-Array Format Manual Protocol. This quick reference document is intended for experienced users.
Beckman Coulter documents
Biomek® Liquid Handler User’s Manual 987834 This document is installed at the same time as the Biomek FXP Target Prep Express software. To access, click Start All Programs Beckman Coulter Manuals.
Biomek® Software User’s Manual B30026AA This document is installed at the same time as the Biomek FXP Target Prep Express software. To access, click Start All Programs Beckman Coulter Manuals.
Table 94 Documents related to the Axiom™ 2.0 Assay 96-Array Format Automated Workflow for Biomek FXP
Document Publication number Description
324 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
Documentation and supportCustomer and technical support
Customer and technical support
Visit thermofisher.com/support for the latest in services and support, including:
• Worldwide contact telephone numbers
• Product support, including:
– Product FAQs
– Software, patches, and updates
• Order and web support
• Product documentation, including:
– User guides, manuals, and protocols
– Certificates of Analysis
– Safety Data Sheets (SDSs; also known as MSDSs)
Note: For SDSs for reagents and chemicals from other manufacturers, contact the manufacturer.
Limited product warranty
Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies’ General Terms and Conditions of Sale found on Life Technologies’ website at www.thermofisher.com/us/en/home/global/terms-and-conditions.html. If you have any questions, please contact Life Technologies at thermofisher.com/support.
Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP 325
References
Hindorff LA, Junkins HA, Mehta JP, and Manolio TA. A Catalog of Published Genome-Wide Association Studies. Available at: www.genome.gov/gwastudies. Accessed 09/28/2009.
Klein RJ, Zeiss C, Chew EY, et al. Complement factor H polymorphism in age-related macular degeneration. Science 2005, 308:385–89
Manolio T.A. and Collins F.S. The HapMap and Genome-Wide Association Studies in Diagnosis and Therapy. Annu Rev Medicine 2009, 60:443–56
326 Axiom™ 2.0 Assay 96-Array Format Automated Workflow User Guide—Biomek FXP
For support visit thermofisher.com/support or email techsupport@lifetech.com
thermofisher.com
04 September 2018