4. After aspirating PBS, add 30ul of pre-warmed trypsin. Be sure to cover the entire plate by carefully swirling it.
5. Incubate in a 37˚C incubator for 5 minutes and check under the microscope. If you see big clumps of cells, incubate additional 2-5 minutes. Do not over-trypsinize cells.
6. While cells are incubating, remove gelatin from the pre-treated plates by quickly flicking gelatin off the plates. Carefully label the colony numbers on all three plates identically. Two of the new plates will be frozen, and the other one will be used to extract DNA to genotype.
7. Once majority of the cells are uniformly dispersed into small clumps or single cells, inactivate trypsin by adding 150ul of medium. Gently pipette up and down using P200 Rainin multichannel pipette 10-15 times without making bubbles.
Transfer 50 ul of cell suspension to each plate. Add 150ul of additional medium to bring the final volume to 200ul.
8. Change media everyday until the cells are ready to be frozen. This will take 2-4 days depending on confluence of the original master plate.