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Tuesday/Wednesday, Day 14/15
Examine the 24 well plates under the scope. You should see a single clone in each well. To disaggregate each clone, hold the dish at an angle and suction out the media. Now add 0.5 ml PBS to each well to rinse out the media and resuction each well. Next add 0.2 ml Trypsin/EDTA to each well and place the plate back in the incubator for 20 minutes at 37oC. Finally add 1.0 ml ES selection media to each well and pipette the cells up and down 4 or 5 times with a 1000ul barrier tip to disaggregate the clone . After all clones are disaggregated, return your 24 well plate to the incubator. Look at your plates every day. You want your wells to have many small nests of colonies, evenly dispersed throughout the well. Note: ES selection media should be used until the clones are frozen to ensure that no Wild Type ES cells contaminate your clones.
Wednesday/Thursday, Day 15/16
Examine the clones with the inverted scope. They will look like many tiny clones of ES cells, evenly dispersed. Aspirate the existing media from the cells, then feed all your wells with 1.0ml ES selection media.
Thursday/Friday, Day 16/17
The individual wells of the 24-well plate should contain 50 to 100 small, healthy, undifferentiated colonies if they were properly disaggregated. Number your wells from 1 to 120. To freeze the individual clones, first rinse each well with 0.5 ml PBS, and then add 0.2 ml 0.05% Trypsin/EDTA to each well. Place the plate in the incubator for 15 minutes. Remove the plate from the incubator and add 0.5 ml ES media to each well. Individually disaggregate the cells with a 200 to 1000 ul barrier tip by pipetting up and down 4 to 5 times. Place 500 ul of the disaggregated colonies in the Nunc vial (1.8 ml) that is numbered the same as your well. Add 0.5 ml of 2X ES freezing media to each vial. Place the numbered cryotubes in a freezer box labeled with your constructs name and put in a -70o freezer. After removing all the colonies to the cryotubes, refeed each well of the 24 well plate with 1.0 ml ES media. Allow the cells in these wells to grow to confluence (which will take 4-6 days) and use these wells to harvest cells for DNA analysis of the clones. Repeat this freezing procedure for all of your plates. When the DNA analysis confirms which numbered vials are homologous recombinants, these vials are then stored under liquid nitrogen for later expansion and injection. If all of your wells were not ready to be frozen by Friday, you may need to disaggregate these wells again with Trypsin / EDTA . This will enable a well containing a small number of cells to be expanded further for a subsequent freeze on Sunday. Follow the freezing procedure above on Sunday for these wells.
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