This protocol can be used to freeze down your 96-well plate for future expansion of your positive clones. This protocol is very useful because it allows you to characterize your clones and not have all of the clones up in culture.
Freeze the plates when colonies are beginning to get 50-80% confluent.
1. Feed plates with NO DRUGS 2-3 hours before freezing.
2. Aspirate media, wash with PBS.
3. Add 30 ul of trypsin and incubate for 5 min.at 37C.
4
. Inactivate the trypsin with 50 ul of FBS.
5. Break up the colonies by pipetting 4-5X.
6. Add 50 ul of 20% DMSO and 80% FBS.
7. Wrap plates in Saran wrap and place carefully in a Styrofoam box.
8
. Place box in -80 freezer.
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