Preparing E8.5 Embryos for Karyotyping

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

Day 1: Culturing Embryos

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

Remove media.
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
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.
Immediately add 1 ml media.
Pipette up and down to get into single cell suspension and transfer to Eppendorf.
Spin down at no more than 500 g for 2 minutes.
Pipet off media and add 500 µl 0.56% KCl and incubate at 37°C for 20 minutes.
Spin down at no more than 500 g for 2 minutes.
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.
Add 1 ml fixative -- Methanol:Acetic Acid (3:1) -- and place at 4°C for 20 minutes.
Spin down at no more than 500 g for 2 minutes.
Add 200 µl fresh fixative [Methanol:Acetic Acid (3:1)].
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.