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HOME > Protocols > Molecular Biology > Cloning > Protocol for E. coli Competent Cell Preparation

Protocol for E. coli Competent Cell Preparation

  1. Inoculate 5ml of L-broth with a single colony of selected E. coli strain and incubate overnight at 37oC. with moderate agitation(~250rpm)
  2. Inoculate 50ml of L-broth with ~100-300ml of the overnight culture and incubate at 37oC. with moderate agitiation(~250rpm) until the A595=0.375 ( initial inoculum should have A595<0.1)
  3. Place culture on ice in autoclaved, prechilled Corex tubes for 10 minutes
  4. Centrifuge cells 7 minutes at 2500rpm(+4oC) and then resuspend pellet (very gently by hand-do not vortex) in 10ml CaCl2 solution( 60mM CaCl2, 15% glycerol, 10mM PIPES, pH 7.0)
  5. Centrifuge cells 5 minutes at 2500rpm(+4oC) and then resuspend pellet (very gently by hand-do not vortex) in 10ml CaCl2 solution
  6. Leave cells on ice for 30 minutes and then centrifuge for 5 minutes at 2500rpm(+4oC) and resuspend cells (very gently by hand-do not vortex)in 2ml CaCl2 solution
  7. Aliquot final preparation in 100ml aliquots on dry ice and store at -70oC. (cells should be viable for up to a year at -70oC)
L-broth: 1% bactotryptone, 1% NaCl, 0.5% yeast extract


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