Authors: Constance Jeffery ### Abstract Simple directions to prepare frozen permanents of E. coli or other bacterial cultures that can be stored for years at -80°C. ### Introduction 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. ### Reagents 1. 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 - LB - Ampicillin or other appropriate antibiotic - Sterile test tubes - Roller - Ice bucket with dry ice - Box for -80°C freezer ### Equipment -80°C freezer ### Procedure 1. 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. ### Timing 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](http://dx.doi.org/10.3791/2134) ### Author information **Constance Jeffery**, Connie Jeffery's Lab Correspondence to: Constance Jeffery (cjeffery@uic.edu) *Source: [Protocol Exchange](http://www.nature.com/protocolexchange/protocols/2120) (2011) doi:10.1038/protex.2011.228. Originally published online 14 June 2011*.