Chemically Competent E.coli Cells
1. Inoculate 100ml of medium A with single colony or 0.5ml culture.
2. Grow to mid log phase @37 (OD(600)=0.4-0.8)
3. Keep on ice for 10 minutes
4. Pellet cells at 1500 x g for 10 minutes at 4 C.
5. Resuspend pellet in 1.0 ml of medium A precooled on ice.
6. Then add 5.0 ml of storage solution B.
7. Divide into aliquots of 0.10 ml each and store in -70 c
(quick freeze is optional)
Medium A: LB supplemented with 10 mM MgSO4-7H2O and 0.2% Glucose.
Medium B: 36% glycerol, 12% PEG (MW7500), 12 mM MgSO4-7H2O added
to LB and sterilized through filtration.
Nishimura, A.,Morita, A., Nishimura, Y., and Sugino, Y. A rapid and highly efficient method for preparation of competent Escherichia coli cells. (1990) Nucleic Acid Res 18:20, p 6169.
Made Feb. 22, 1999; Baker Laboratory
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