Nucleic Acid Prep Station

What we can do for you!

The ABI 6100 PrepStation

• Isolates and purifies total RNA and genomic DNA

• High throughput, up to 96 wells at a time.

• Isolates high purity nucleic acids free of cross contamination.

Genomic DNA System

• Provides DNA isolation from cultured cells, whole blood, buccal swabs, plant and animal tissues.

• The isolated DNA from blood is free from contaminating protein such as heme and other cellular macromolecules.

• Provides high molecular weight genomic DNA free from inhibitors of PCR and suitable for any DNA based assay application.

Total RNA System

• Produces high quality total RNA from cultured cells, whole blood, plant and animal tissue.

• Produces inhibitor free RNA for Real Time PCR or microarray analysis.

• Absolute RNA wash minimizes genomic DNA content.

Operational Policies

• Fill out an order form on the biodesktop. https://biodesktop.uvm.edu/

Operational Policies continued

• Drop off samples at the DNA Facility HSRF 305.

• Blood samples should be frozen.

• Cultured cells should be dried down and frozen.

• Tissue samples should also be frozen.

gDNA Results

• DNA yields from tissue culture cell lines vary depending on the ploidy (number of chromosomes per cell) of the cell line.

• The purification

process recovers gDNA linearly for input ranges of 10-106cells.

 

SAMPLE TYPE

INPUT VOLUME (ul) YIELD (µg)

PURITY(A260/280)

Human Blood 150 3-8 1.7-1.8

Rat Blood 50 3-5 1.7-1.8

Mouse Blood 50 3-5 1.7-1.8

SAMPLE TYPEINPUT # OF CELLS YIELD (µg)

PURITY (A260/280)

HeLa Cells 105 12 1.8

PC3 106-107 8 1.8

Raji 106-107 5-6 1.8

L1210 106-107 2-5 1.8

Total RNA Results

• The Total RNA chemistry produces RNA that contains less than 0.5% gDNA contamination by weight. It also removes virtually all protein, providing a highly stable total RNA.

•  The input range for cells is 10 to 106, but the recommended range is 104 to 106 cells per well. The total RNA recovery versus cell input is linear.

18

S

28

S

Flu

or

es

ce

nc

e

T ime (seconds)

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

19 24 29 34 39 44 49 54 59 64 69

The DNA Analysis Facility isolated total RNA that was tested for degradation. Ribosomal RNA [28s/18s] peaks indicate fully intact RNA. Provided by Agilent 2100 Bioanalyzer.

Quality Control:

What the facility will do:Nanodrop Spec. Analysis

-Report concentration in ng/ul-260/280 ratio to determine purity-260/230 ratio to assess level of residual salts

RNA Integrity Analysis:-Gel or Bioanalyzer

What we suggest you do:Check level of gDNA via PCR

Expectations:

• Nucleic Acid concentrations– gDNA:

• Whole Blood: 40ng/ul• Tissue: 35-125 ng/ul• Cells: 20-190ng/ul

– RNA:• Tissue:5-125 ng/ul • Cells: 30-78ng/ul

• Requires 100-150ul elution volumes• Turn around 1-3 days depending on sample type• Cost: $3.91/well

Conclusions

• This approach is best suited for blood, cells and normal tissue. Not well suited for small recovery samples.

• Future Considerations: Develop better approaches for difficult and low recovery samples.

• Contact Meghan in the DNA Facility, 6-2557.