Microbiology Biochemistry

scientificprotocols authored over 7 years ago

Authors: Constance Jeffery


Simple directions to prepare frozen permanents of E. coli or other bacterial cultures that can be stored for years at -80°C.


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.


  1. 100% w/v glycerol sterile filtered (per 100ml: weigh 100g. of glycerol, add water to bring volume to 100ml. Then sterile filter)
  2. Sterile screw top freezer tubes
  3. LB
  4. Ampicillin or other appropriate antibiotic
  5. Sterile test tubes
  6. Roller
  7. Ice bucket with dry ice
  8. Box for -80°C freezer


-80°C freezer


  1. Inoculate 5ml LB 100ug/ml ampicillin (or other appropriate antibiotic for your culture) with cells.
  2. Grow overnight on roller.
  3. Mix 800ul culture with 200ul 100% w/v glycerol (sterile) in sterile screw cap freezer tube. (usually make several tubes)
  4. Invert a few times
  5. Bury tube in dry ice until frozen solid.
  6. Store in -80°C freezer.


Overnight to grow culture plus about 15 minutes to complete the rest of the protocol.

Anticipated Results

Frozen permanents should be good for years if stored at -80°C.

Associated Publications

Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein. Forum H. Bhatt and Constance J. Jeffery. Journal of Visualized Experiments 03/12/2010 doi:10.3791/2134

Author information

Constance Jeffery, Connie Jeffery's Lab

Correspondence to: Constance Jeffery ([email protected])

Source: Protocol Exchange (2011) doi:10.1038/protex.2011.228. Originally published online 14 June 2011.

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