Nucleic Acid Prep Station

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Nucleic Acid Prep Station

• What we can do for you!

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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.

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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.

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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.

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Operational Policies

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

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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.

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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.
•  

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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.

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.
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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

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Expectations

• Nucleic Acid concentrations
• gDNA
• Whole Blood 40ng/ul
• Tissue 35-125 ng/ul
• Cells 20-190ng/ul
• RNA
• Tissue5-125 ng/ul
• Cells 30-78ng/ul
• Requires 100-150ul elution volumes
• Turn around 1-3 days depending on sample type
• Cost 3.91/well

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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.