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 37^oC. with moderate agitation(~250rpm)
   
2. Inoculate 50ml of L-broth with ~100-300ml of the overnight culture and incubate at 37^oC. 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(+4^oC) 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(+4^oC) 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(+4^oC) 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 -70^oC. (cells should be viable for up to a year at -70^oC)

L-broth: 1% bactotryptone, 1% NaCl, 0.5% yeast extract