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Latest revision as of 19:54, 19 October 2016

Protocol 11 : Glycerol storage

Aim: Safely store cells at -80°C

Following steps will be performed under a class II PSM

Glycerol Freezing Solution (50%)

Prepare a 50% glycerol stock solution by mixing 5mL glycerol with 5mL distilled, sterile water.
Mix well to make sure that you see one uniform solution, and there are no layers present.

Bacteria storage

Mix 50µL of the overnight culture of bacteria of interest with 50µL 50% glycerol solution (25% glycerol final concentration) into a 2mL cryotube
NB : Snap top tubes are not recommended as they can open unexpectedly at -80°C
Label both the lid and the tube of the cryotube. Frozen tubes are hard to write on and samples stored for long periods at -80°C can lose labels stuck to tube. Store at -80°C

Bacteria recovery

Pipet bacteria cells and put it into a 500mL sterile Erlenmeyer containing 100mL of sterile liquid LB medium. Cover with aluminum paper and place the Erlenmeyer in a rotative incubator at 37 Celsius degrees, 120rpm O/N.

Sources

  • Addgene protocol for creating a Glycerol stock

  • Sigma-Aldrich protocol for E.coli manipulations