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Protocol for prepping ES cells for the rodent PCR panel by Charles Rivers Laboratories for PCR viral detection and mycoplasma testing.


Category
Protocol
Record History
Added on February 3, 2012 at 12:56 PM by Skelton, Jennifer
Modified on February 3, 2012 at 1:00 PM by Skelton, Jennifer
Shared with (contributions)
TMESCSR: Protocols Master
  1. Plate an appropriate number of cells on a gelatinized 60 mm dish and grow until confluent, feeding daily.  Cells should be passaged without antibiotics prior to submission.
  2. Feed with cES cell media 1 – 2 hours prior to trypsinization.
  3. Rinse 2 times with 5 ml PBS.
  4. Add 2 ml ES cell trypsin.
  5. Incubate at 37°C for 3 - 5 minutes.
  6. Counter with 4 ml cES cell media.
  7. Pipette up and down until colonies are broken into single cell suspension.
  8. Transfer total volume of cells to a 15 ml conical tube.
  9. Count cells.  Cell count is not critical, but it must be noted if there are more than 5 x 107 cells/ml.
  10. Centrifuge at 1000 rpm for 5 minutes. Aspirate media.
  11. Re-suspend pellet in 500 µl PBS.
  12. Split volume between two 1.5 ml eppendorf tubes.
  13. Label with “Rodent PCR Panel”, Investigator, Construct, Clone, Passage Number, Date.
  14. Store at -80°C.
  15. Contact DAC Comparative Pathology Lab and arrange for a time to drop off the cell samples.

Anne Pate

anne.f.pate@vanderbilt.edu

936-2654

MCN T322

Vials will need to be delivered on dry ice after making arrangements for submission.