Protocol for E. coli Competent Cell Preparation

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Protocol for E. coli Competent Cell Preparation

• Plate the antibiotic free plate with competent cells.

• Inoculate 5ml of L-broth with a single colony of selected E. coli strain and incubate overnight at 37°C. with moderate agitation(~250rpm)

• Inoculate 50ml of L-broth with ~100-300ul of the overnight culture and incubate at 37oC. with moderate agitiation(~250rpm) until the A595=0.375 ( initial inoculum should have A595<0.1)

• Place culture on ice in autoclaved, prechilled tubes for 10 minutes

• 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)

• Centrifuge cells 5 minutes at 2500rpm(+4oC) and then resuspend pellet (very gently by hand-do not vortex) in 10ml CaCl2 solution

• 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

• Aliquot final preparation in 100ml aliquots on dry ice and store at -70oC. (cells should be viable for up to a year at -70oC)

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