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Pharmacology & Toxicology Core Facilities SOP #: 18654.B446B.001 Version #:

Author: Brian Jespersen Page of 10 Approval Date:

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Core Equipment ID: 18654

Description: Applied BioSystems, 7500 Real Time PCR System

Room: B446B (Molecular & Biochemical Core)

Champion:

1.0 Purpose Standardize the process of control, maintenance and ownership, of the Applied BioSystems, 7500 Real‐Time PCR System located in Room B‐446B Life Sciences.

1.1 Applied BioSystems, 7500 RealTime PCR System Capabilities1 Is a powerful platform for labs requiring superior performance and maximum dye versatility. The 7500 Real‐Time PCR System is a sophisticated platform for users who require extended capabilities and maximum versatility. The 3rd generation platform features an innovative optical system that enhances sensitivity and lets you access a broader range of fluorophores. The variable excitation capability allows greater sensitivity for longer wavelength (red) dyes.

a. Powerful, five‐color platform is calibrated for the broadest

range of dyes available: FAM™/SYBR® Green I, VIC®/JOE™, NED™/ TAMRA™/ Cy3™, ROX™/Texas Red®, and Cy5™ dyes.

b. Specialized optical system enables easy and accurate calibration to new dyes without requiring the addition of new filter sets.

c. Advanced multi‐componenting algorithm minimizes spectral

crosstalk – superior for multiplexing.

d. User‐friendly software includes plate set‐up wizards, multi‐plate data viewing capabilities, and advanced analysis tools to make data processing simple and straightforward.

1.2 7500 Software v2.0, Software Capabilities2

17500 Real‐Time PCR System. Applied Biosystems. 25 August, 2008. https://products.appliedbiosystems.com:443/ab/en/US/adirect/ab?cmd=catNavigate2&catID=601251&tab=DetailInfo. 2 7500 Real‐Time PCR System. Applied Biosystems. 25 August, 2008. https://products.appliedbiosystems.com:443/ab/en/US/adirect/ab?cmd=catNavigate2&catID=601251&tab=DetailInfo.



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Now the easy‐to‐use StepOne software is available for the 7500 and with the 7500 Software v2.0 upgrade. The 7500 Software v2.0 incorporates your favorite StepOne Software features, such as a variety of plate setup wizards, standard curve dilution and master mix recipe calculators, QC flags, data filters, and email notification when a run is finished. The 7500 Software v2.0 also includes an enhanced Gene Expression Study package and has a variety of new melting curve protocol options, including multiple peak detection, step and hold temperature control, and customizable ramp rates. The NEW Gene Expression Study package accommodates large studies better than any other instrument software package:

a. Import an unlimited number of Comparative CT (relative quantitation) files to one study.

b. Group samples and view data both by technical replicate group and biological replicate group.

c. Use any gene(s) as an endogenous control, including averaging

multiple controls together.

d. Enter known efficiency values to be factored into the RQ results.

2.0 Reason for Issue

Maintain a document that describes the Standard Operating Procedures that allows for the standard safe and maximal use of the Applied BioSystems, Real Time PCR System within the Pharmacology and Toxicology Core Facilities.

3.0 Process Description Allow Core Facility Users within the Pharmacology and Toxicology Department to properly and effectively use the Applied BioSystems, Real‐Time PCR System. The process description details the standard use of the Applied BioSystems, 7500 Real‐Time PCR System. The controlled standard must maintain and adhere to proper and approved research and regulatory qualitative conditions. 3.1 SOP: 18654.B4446B.001 for Applied BioSystems, 7500 Real Time PCR

System, authored by Brian Jespersen, created on 8/20/2008, issued on 8/25/2008.

3.2 SOP: 18654.B446B.001 applies to any User and / or Trainer of the Applied BioSystems, 7500 Real Time PCR System.



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3.3 Responsibilities

All Users are responsible for obtaining the proper approval and training before the use of the Applied BioSystems, 7500 Real Time PCR System. All Users are responsible for the proper use, according to defined protocol, when using the Applied BioSystems, 7500 Real Time a. New Users need a new Applied BioSystems, Real 7500 Time PCR

System account created for equipment access, before initial use. New accounts are authorized and created by the Equipment Champion and / or the Core Facility Manager. A new account maybe created after training and equipment approval has occurred. New accounts must turn off the default computer sleep mode. (Under: Start/Control Panel/Display Settings/Screen Saver/Monitor Power (Power)/Power Schemes) Select “Always On.” Click “Ok” and “Ok” again. This process disables the default system “sleep” mode. If the computer enters sleep mode, Applied BioSystems, 7500 Real Time PCR System data may be lost.

b. All Users must schedule equipment use in the Equipment Schedule Logbook prior to use. The Equipment Schedule Logbook is located on the desk next to the Applied BioSystems, 7500 Real Time PCR System.

c. All Users must record all equipment use in the Equipment Usage Logbook post‐use on the same day as the recorded use. The Logbook is located on the desk next to the Applied BioSystems, 7500 Real Time PCR System. Within the Logbook on the current log sheet, Users must record the following: Date, PI, Name, Lab Location, Phone Number, Program, Samples, Cycles, Error Messages.

d. Only covered samples may enter Room B‐446B.

e. Immediately after use, the Applied BioSystems, 7500 Real Time

PCR System must be turned off to preserve lamp life.

f. Do not close the door to room B‐446B.

g. When not occupying room B‐446B room light off should be off.

3.4 Equipment Safety Issues



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a. Contamination of the thermocycler block can result in high background. To avoid contamination follow the below precautions:

i. Do not open tubes in room. ii. Do not write on tubes – ink will transfer to block. iii. Do not setup reactions with tubes in ice. iv. Do not handle tubes or caps with bare hands. v. Tubes should only have contact with powder‐free

gloves and clean tube rack

b. Instrument Issues: Plates and Tubes: Only ABI optical plates, or tubes and caps maybe used. Caps must be the flat style. Domed caps will cause damage to the machine. Do not close the door to room B‐446B.

3.5 Quality Measures a. Weekly: Once a week the Applied BioSystems, 7500 Real Time

PCR System computer must be shut down and rebooted.

b. Monthly: Once a month a Background Calibration and Optical Calibration must be preformed on the Applied BioSystems, 7500 Real Time PCR System. Use the Applied Biosystems Instillation and Maintenance Guide (location of guide see section 3.10) to perform these monthly quality assurance measures. Once these calibrations have been completed, the date, time and person who performed the calibrations, must be recorded in the Quality Measures Logbook.

c. Every Six Months: Once every six months a Region of Interest calibration and a Pure Dye Calibration must be preformed on the Applied BioSystems, 7500 Real Time PCR System. Use the Applied Biosystems Instillation and Maintenance Guide (location of guide see section 3.10) to perform these monthly quality assurance measures. Once these calibrations have been completed, the date, time and person who preformed the calibrations, must be recorded in the Quality Measures Logbook.

3.6 Procedure; Applied BioSystems, 7500 RealTime PCR System Use

Setup

a. Log into computer. Sign in under User Account Name and

password. User accounts are sectioned by Laboratory Names.



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b. Click on ABI 7500 Software desktop icon to start use. This software runs the Applied BioSystems, 7500 Real Time PCR System.

c. Within the ABI 7500 Software, click on the “New Document” icon

in the left hand corner of the screen.

d. Define the document as either Absolute or Standard / Relative Quantization.

e. Choose Plate – 96 clear well plate and enter a plate name. Click

“Next” button.

f. Choose detectors, the gene of interest being analyzed. Select the detector and add it to the document. This must be done for every well on the plate. Click “Next” button.

g. The plate setup screen will appear. Assign each well a label.

h. Highlight the box next to the well display on the screen. Assign

individual well task and enter the quantity of DNA in the quantity sample box for all wells. Make sure to properly label each well. ie; Unknown, Control, Standard, etc.

i. Turn the Instrument ON. This must be done before pressing

the “Finish” button or the computer cannot detect the instrument. The Applied BioSystems, 7500 Real Time PCR System ON button is located on the front of the system.

j. When all well parameters are defined, click “Finish.” The computer will detect the Applied BioSystems, 7500 Real Time PCR System. Noise from the Applied BioSystems, 7500 Real Time PCR System should be heard at this point.

k. In the ABI 7500 software the 96 Well Template Screen will now open. Under the “Setup” tab, individual wells / samples can now be labeled. Multiple wells / samples can be labeled by highlighting multiple boxes at once.

l. The default analysis settings can be found under the “Instrument”

tab. These settings may be modified prior to use. The default settings are: Sample Volume 50 µL, Stage 3, Step 2, 60 °C, 1 minute. An important default deviation is the addition of the dissociation stage. This stage is necessary when testing primers for the first time; assay validation is achieved. The data collection time point can also be changed.



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m. Save the document as a *.spd file in the proper corresponding User

Laboratory folder.

Sample Preparation n. Samples that require centrifuging must follow the steps under

Sample Preparation. If centrifuging is not required proceed to the Sample Analysis section.

o. To centrifuge samples, use the Eppendorf Centrifuge 8504, which

is located in room B‐446B on the desk adjacent the Applied BioSystems, 7500 Real Time PCR System. 96 well plates and individual sample tubes may be centrifuged with the Eppendorf Centrifuge 8504.

p. To turn on the Eppendorf Centrifuge 8504 by pressing the “On”

button.

q. Press “Open” to open the Eppendorf Centrifuge 8504 access door. The Eppendorf Centrifuge 8504 will unlock and the top access door may be lifted open.

r. Securely place samples in the proper tray. Two trays are available,

a 96 well plate tray and an individual sample tube tray. To switch between the two trays, use the Allen Wrench, located next to the Eppendorf Centrifuge 8504, and remove the center bolt. Lift the tray out and replace with required tray. Retighten the bolt with the Allen Wrench.

s. Sample must have a counterbalance. The 96 well plate

counterbalance is located with the 96 well plate Eppendorf Centrifuge 8504 tray. Individual sample tube counterbalances are the responsibility of the User. Both samples and counterbalances must be secured and covered.

t. Close the lid of the Eppendorf Centrifuge 8504. Set preferred

centrifuge speed and time.

u. Press the “Start” button to activate the centrifuge. Wait until the Eppendorf Centrifuge 8504 has stopped before opening sample access door. Do not force the Eppendorf Centrifuge 8504 sample access door open.

v. All Eppendorf Centrifuge 8504 use must be recorded in the

Eppendorf Centrifuge 8504 Logbook, which is located next to the



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Eppendorf Centrifuge 8504. Users must record the following on the current Logbook form: Date, PI, Name, Lab Location, Phone, Rotor, Time, Number, and Problems / Comments.

w. Eppendorf Centrifuge 8504 manuals and user guides are located in

the second, left desk door.

Sample Analysis

x. Open the door to the Applied BioSystems, 7500 Real Time PCR System by pressing the circular indent on the front of the machine.

y. Insert the well plate in the aligned position. Make sure to use the

proper plate; either 96 well plate or the 8 strip‐tube plate for individual tubes. Make sure the plate is properly inserted and secured.

z. Close the Applied BioSystems, 7500 Real Time PCR System door

by pushing on the circular indent located on the front of the open door until the door is securely closed.

aa. When the 96 well plate settings are properly defined, click “Start”

in the ABI 7500 software. Once started, the ABI 7500 Software will estimate the total read time. Once the proper temperature is reached the Applied BioSystems, 7500 Real Time PCR System will start.

bb. The ABI 7500 Software, under the “Methods” Section, will in real‐

time monitor the block, heated cover, sample temperature, repetition and display the current cycle location. At this point previous data may be manipulated while the system performs the selected task.

cc. When finished with the Applied BioSystems, 7500 Real Time PCR

System, close all programs and completely log off the computer.

dd. Make sure Applied BioSystems, 7500 Real Time PCR System is off.

3.7 Core Materials

Currently, there are no Core Materials provided for the Applied BioSystems, 7500 Real Time PCR System.

3.8 User Materials Applied BioSystems, 7500 Real Time PCR System User Materials maybe located and purchased from the below web addresses. The



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available materials include, but are not limited to: Assays, Buffers, Enzymes, Master Mixes, Primers, PCR Controls, Reagents, Services, Thermal Cyclers, Trays, Tubes, Etc. These materials will allow for the proper and optimal use of the Applied BioSystems, 7500 Real Time PCR System. Users are individually responsible for the compatibility, maintenance and purchase of these materials. Related Products https://products.appliedbiosystems.com:443/ab/en/US/adirect/ab?cmd=catNavigate2&catID=601251&tab=RelatedProd. PCR / RTPCR Products https://products.appliedbiosystems.com/ab/en/US/adirect/ab;jsessionid=GXtCLySTsPdBh1mTX8cCprJybWVbZvv92GHFcGMp0LQ4zPz0f5fG!1185967524?cmd=catNavigate2&catID=600961.

3.9 User Use Records a. Records of Use All Applied BioSystems, 7500 Real Time PCR

System use must be recorded. Refer to 3.3b, 3.3c.

b. Error Messages / System Issues All error messages and system issues must be relayed to the Equipment Champion and the Pharmacology & Toxicology Core Facility Manager. This information must be relayed on the same day as equipment use. Error messages / system issues must be recorded, refer to 3.3c.

3.10 Resource Index

Applied BioSystems, 7500 Real Time PCR System literature and resources for the follow items can be found at the link below. Printed versions of these resources can also be found in the top left desk door adjacent the Applied BioSystems, 7500 Real Time PCR System in room B‐446B.

a. Brochure. b. Installation and Maintenance Guide c. Starting Guide: Absolute Quantitation Using Standard Curve. d. Starting Guide: Allelic Discrimination Experiments e. Starting Guide: Plus / Minus Assay Experiments f. Starting Guide: Relative Quantitation Using Comparative CT g. Computer Setup h. Specification Sheet i. User Bulletins j. Data Comparison Guide k. Etc.



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https://products.appliedbiosystems.com:443/ab/en/US/adirect/ab?cmd=catNavigate2&catID=601251&tab=Literature Applied Biosystems Company Representatives Field Service Engineer Devin Millions [email protected] (800) 831‐6844 Customer Support / Sr. Field Application Specialist Chris Miller [email protected] (800) 248‐0281 x7611

4.0 Competences, Authorization and Training New Users must receive proper authorization from either the Equipment Champion and / or Pharmacology & Toxicology Core Facility Manager before equipment use. A new User may contact the Equipment Champion or Pharmacology & Toxicology Core Facility Manager to schedule training. Training includes SOP and instrument familiarization and any additional required or specialized training. Once training is complete authorization may be issued and a system account and password may be setup. All Users are individually responsible for current SOP familiarization. All New Users must refer to 3.3a during new Applied BioSystems, 7500 Real‐Time PCR System account creation.

5.0 SOP Performance and Equipment Review

The effectiveness of the SOP: 18654.B4446B.001 will be monitored by the Pharmacology & Toxicology Core Facility Manager, Equipment Champion and All Users. Any procedural or qualitative deviations will be reflected within an updated SOP. Any Approved User should aptly report any procedural or qualitative issues and / or errors to the Pharmacology & Toxicology Core Facility Manager or Equipment Champion. The Equipment Champion’s name and contact information can be found on the Core Facility Identification Sticker. The Core Facility Identification Sticker is located on the Applied BioSystems, 7500 Real‐Time PCR System. The Core Facility Manager’s contact information maybe found on the Core Facility Contact Posting within room B‐446. Updated SOPs will be published and Approved Users will be notified. SOP: 18654.B4446B.001 review will occur every two years. 5.1 Update Date:

Reason for SOP Change: New Version #:



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5.2 Update Date

Reason for SOP Change New Version #:

6.0 Definitions

a. SOP Standard Operating Procedure, which is a standard guide that officially standardize the process of control, maintenance and ownership of the Applied BioSystems, 7500 Real‐Time PCR System. The SOP number stand for (xxx . xxx . xxx) equipment number . room number . SOP version number. b. Originator / Author The individual representing the Pharmacology and Toxicology Core Facility that created SOP: 18654.B446B.001. c. Stakeholder Any individual that uses the Applied BioSystems, 7500 Real‐Time PCR System, including the Pharmacology and Toxicology Core Facility Department. d. New User An individual who has not completed the requirements of section 4.0. e. Approved User An individual who uses the Applied BioSystems, 7500 Real‐Time PCR System and has fulfilled section 4.0. This title may only be given by the Equipment Champion and / or the Pharmacology and Toxicology Core Facility Manager. f. Champion An individual who’s direct expertise with the Applied BioSystems, 7500 Real‐Time PCR System has been recognized by the Pharmacology and Toxicology Core Facility Committee. This title may only be awarded by the Pharmacology and Toxicology Core Facility Committee.

7.0 Appendix This SOP was written by: Brian Jespersen ___________________________________

This SOP was reviewed by: Dr. Stephanie Watts ______________________________ This SOP was authorized by: Dr. J.R. Haywood __________________________________ Issue Date: _____________________________________

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