About 7~8 days after drug selection is started, colonies are big enough to be picked with naked eyes. Once you know your general targeting efficiency, you may adjust the numbers to pick. We pick 48 colonies per construct.
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Materials:
round-bottom 96 well plate
flat-bottom gelatin-coated 96 well plate
Methods:
- Aliqot 10ul of trypsin per well to a 96-well round bottom plate.
- Prepare a ES cell plate by rinsing with 10ml of PBS and add 2ml of PBS to cover the plate.
Place the plate under the microscope to select undifferentiated healthy colonies. Pick colonies using a P-20 pipette set to 10ul. Carefully pick a colony without breaking up the colony into single cells, and transfer it into a 96-well plate with trypsin. Pipette up and down a couple of times to disperse the colony. Once all 48 colonies have been picked into the wells, incubate the plate in a 37˚C incubator for 7-10 minutes. - Add 150ul of media to each well. Pipette up and down ~10-15 times using the multichannel pipette to break up the colonies.
Transfer the cell suspension to a 96-well gelatinized plate. Change media everyday until ready to be split again.
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