See above for notes on splitting ES cells, frozen. Generally, ES cells are split 1:6-1:10. This depends on how "confluent" the plate is. Since ES cells grow as discrete colonies, you should NEVER get a plate that is confluent in ES cells the way fibroblasts are. You wish to split them when a colony is approximately 1-2 times the diameter of a stretched out fibroblast (STO cell). If the plate is densely covered with colonies, they may be split 1:10, or slightly greater. If there are a lot of colonies, split them 1:6-1:8. For a plate that has only a few colonies (12-20), allow them to get slightly larger than normal, and then split 1:4. To split, simply add 1ml. of the appropriate dilution of cells to a 100cm. plate containing a feeder layer, and 4 ml. of ES cell media.

When ES cells are "confluent" and ready to split (see above) feed them with fresh media an hour or two before splitting. Aspirate media and wash cells once briefly with 1X trypsin-EDTA, aspirate. Add 0.5ml 1X trypsin-EDTA (for one well of a 6-well, 2 ml. for a 100cm plate). Then, add .5 ml. pre-warmed (37°C) trypsin to a 6-well, or 3ml. to a 100cm. and put at 37°C for 5-6 min. You wish to minimize this time, so monitor the cells carefully. When cells are loosened add 0.5ml ES cell media to the well with a 5 ml pipette. Attach a sterile yellow tip to the end of a 10 ml sterile plactic pipette and pipette cells up and down several times -- ES cells are sticky and this is necessary to achieve a single-cell suspension to prevent differentiation after plating. For a 100cm. plate, transfer the cells to a Centrifuge tube containing an equal volume of media:trypsin. Pipette back and forth at least three times using a sterile yellow tip attached to the bottom of a 10ml. pipette. Transfer cells to a l5ml centrifuge tube, add another 5 ml ES cell media and spin 2 min at 2000 rpm. For a 100cm. plate, add another trypsin volume's worth of ES cell media to the centrifuge tube, and mix. This may require the use of a 50 ml centrifuge tube. Spin 2 minutes at 2000 rpm. Aspirate and resuspend cells in ES cell media, plate onto fresh feeder cells (remember to be careful about the split ratio. ES cells will grow slowly, or not at all, if they are not dense enough. Unless the plate that you are splitting from is extremely dense, split cells no more than 1:10)

Freeze as for STO/SNL cells, ES freezing media is regular ES media with 20% FBS and 10% DMSO. It is very important that the cells be cooled slowly, to prevent the formation of ice-crystals within the cells. This can be accomplished one of two ways. One way, is to freeze the cells at -20°C for an hour, and then transfer them to a -70°C overnight. The next day, move them to liquid nitrogen. An alternate way to freeze cells, if to use a cell-culture cooler (simply a thick styrofoam box, with a few holes poked in it). Place the cells inside the cell-culture cooler, and place at -70°C overnight. The next day, transfer the cells to liquid nitrogen.

Digest 100µg of targeting construct DNA with an enzyme to linearize Check 0.5µl on a gel to make sure it's completely digested. Extract DNA 2X with phenol/chlorofom. Add .15 volumes 2M NaOAc and 2 volumes EtOH to precipitate (the DNA will probably come right out of solution so freezing isn't needed). Pellet the DNA, wash with 70% EtOH and dry the pellet. Resuspend the DNA in 100µl sterile H20 or TE (you should be working in the hood here). Check 0.5 µl DNA on a gel, and make sure that the concentration is know.