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Wednesday, Day 8
Feed transfected ES cells with 13 ml of Selection Media. (G418 [Geneticin, Gibco] @ 600 ug/ml active concentration). Remember to feed your ongoing MEFs with fresh MEF media.
Thursday, Day 9
Feed the transfected ES cells with Selection Media, 13 ml dish. The clones should now be fairly large.
Friday, Day 10
Feed transfected ES cells with Selection Media as above. You should begin to see some selection in your dishes. Dead cells should be suspended in the media above your ES clones. Using your ongoing T-150 MEF flasks (2 flasks), you will prepare six, 24 well dishes for the isolation and expansion of your individual clones on Monday. MEF passage 3: split ongoing MEFs (90% confluent) and irradiate as follows: Take off the old media and rinse x 1 with PBS (10 ml). Add 7 ml Trypsin/EDTA and incubate 5 to 10 minutes at 37oC. Add 10 ml MEF media, and pipette up and down. Transfer all of the MEF cells to a 50 ml centrifuge tube. Repeat with the other flask of MEFs. If you wish to keep an ongoing flask of MEF cells at this time, you may leave 5 ml. of cells in one of the flasks and refeed this flask. However after preparing your 24 well dishes today you will not need MEFs again for the completion of this electroporation. Take the MEF cells in the centrifuge tube and irradiate the cells as before (3000 rads) at the blood bank. Using a hemacytometer, count your MEFs. You will need 7x 104 MEFs per well. To attain this, you should add 9x106 irradiated cells in a final volume of 125 ml. MEF media into a sterile plastic bottle. Now you will have enough cells to prepare 6 - 24 well dishes. Mix the cells gently so that they are evenly dispersed and add 1.0 ml of irradiated MEFs to each well of a 24 well dish in a total of 6 dishes. Incubate at 37oC until Monday.
Sat. or Sun., Day 11/12
Feed transfected ES cells with 12 ml of Selection Media (G418). This may be done on Sat. or Sun., but does not have to be done both days.
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