Simple directions to prepare frozen permanents of E. coli or other bacterial cultures that can be stored for years at -80°C.
Frozen Permanents of E. coli Cultures
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posted 14 Jun, 2011
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This is a simple protocol for preparing frozen permanents of E. coli or other bacterial cells. The frozen permanents can be stored for years at -80°C.
100% w/v glycerol sterile filtered (per 100ml: weigh 100g. of glycerol, add water to bring
volume to 100ml. Then sterile filter)
Sterile screw top freezer tubes
Ampicillin or other appropriate antibiotic
Sterile test tubes
Ice bucket with dry ice
Box for -80°C freezer
Inoculate 5ml LB 100ug/ml ampicillin (or other appropriate antibiotic for your culture) with cells.
Grow overnight on roller.
Mix 800ul culture with 200ul 100% w/v glycerol (sterile) in sterile screw cap freezer tube. (usually make several tubes)
Invert a few times
Bury tube in dry ice until frozen solid.
Store in -80°C freezer.
Overnight to grow culture plus about 15 minutes to complete the rest of the protocol.
Frozen permanents should be good for years if stored at -80°C.