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Preparing E8.5 Embryos for Karyotyping

Pre-gelatinize a 24-well plate and pre-warm media.

Day 1: Culturing Embryos

  1. Harvest embryos (Keep a portion for genotyping.).Transfer embryos into individual wells of a 24-well plate containing sterile PBS.
  2. Move to sterile hood and transfer embryos into Eppendorf tubes with minimal PBS.
  3. Add 50 µl 0.25% trypsin and place in 37°C incubator for 3 minutes.
  4. Immediately add 100 µl media.
  5. Mechanically disrupt embryos with Pipetman and transfer into individual wells containing 0.5 ml media in gelatinized 24-well plate.
  6. Incubate overnight at 37°C with 5% CO2.

Day 2: Fixing Embryos

  1. Remove media.
  2. Add 500 µl fresh pre-warmed media containing 50 ng/ml colcemid.

    2.5 µl of a 20 µg/ml stock per ml of media
  3. Incubate 4 hours.

    Note that time and concentration may vary, so you may want to test ahead of time with a wild-type litter.


    Remove media and add 250 µl 0.25% trypsin and place in 37°C incubator for 3 minutes.
  4. Immediately add 1 ml media.
  5. Pipette up and down to get into single cell suspension and transfer to Eppendorf.
  6. Spin down at no more than 500 g for 2 minutes.
  7. Pipet off media and add 500 µl 0.56% KCl and incubate at 37°C for 20 minutes.
  8. Spin down at no more than 500 g for 2 minutes.
  9. Decant solution. Flick tube to resuspend cells in remaining solution.

    This is important! If you don't do this, all your cells will clump together.
  10. Add 1 ml fixative -- Methanol:Acetic Acid (3:1) -- and place at 4°C for 20 minutes.
  11. Spin down at no more than 500 g for 2 minutes.
  12. Add 200 µl fresh fixative [Methanol:Acetic Acid (3:1)].
  13. Keep at 4°C until ready for spreads.

    Note: When you do spreads, if you see crystallization on the slide, either your Methanol:Acetic Acid solution is bad, or you still have KCl left in it and need to do an extra fix change.

Media

  • 410 ml DMEM
  • 75 ml FBS
  • 5 ml 100X Pen/Strep

100X b-Mercaptoethanol (b-ME)

  • 50 ml 1X PBS
  • 36 µl 14M b-ME

© 2014 The University of Texas MD Anderson Cancer Center