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Protocols: ES Cell Culture - Gene Targeting - Protocols

Picking ES Cell Colonies and
Transferring Them to 96-Well PLATES



	1.	Add 20 - 25 ul of trypsin per well to a 96-well plate, 
round bottom plate.

	2.	After flooding the 10 cm plate with PBS, pick the colony 
from the plate using a Pipetman set at 2 - 4 ul, and transfer it 
into the well with trypsin of 96-well plate (1 colony/well).

  3.   After completing a plate (96 colonies picked),  incubate the 
plate in TC incubator at 37 �C  for 10 - 15 minutes.

	4.	Add 30 - 35 ul of media, M15 per well.   Pipette up and 
down to break up the colonies using the multi-channel pipetor.

	5.	Transfer the ES cell suspension into a 96-well feeder 
plate.   Add an additional 100 ul of fresh M15 media per well.   
Return plates to TC incubator 37� C.

	6.	Alternatively, 50% of the cells may be lysed directly 
for PCR at this point.   Add cell suspension directly to PCR Lysis 
Buffer.