Methods:

One colony of BL21 E.coli cells from LB plate was inoculated into 2 mL LB liquid
medium and was shaken at 37°C overnight. 1 mL overnight cell cuture inoculated into
100 mL LB medium and shaken at 37°C to OD1.0. the culture was chilled for 15min
and 0.1M CaCl2 solution, 0.1M CaCl2 + 15% glycerol were on ice. 2 time 45 mL of
cell culture was centrifuged at 4000rpm for 10 min. The medium was discarded and
the cell was resuspended in 40mL 0.1M CaCl2 cold solution. The cells was kept on ice
for 30min and was centrifuged. The medium was discarded and the cell was
resuspended in 0.5mL of 0.1M CaCl2 + 15% glycerol. 200uL of the cell suspention
was pipetted into sterile 1.5 mL micro-centrifuge tubes and frozen at -70°C freezer.

Question:

1. BL21(DE3) competent cells have an all-purpose strain for high-level protein

expression and easy induction. The BL21(DE3)pLysS competent cells provide tighter
control of protein expression for expression of toxic proteins and are resistant to
chloramphenicol. When used with the CE6 bacteriophage, the BL21 cells provide the
tightest control of protein expression.
2. Preparation of bacterial cultures, growth of electrocompetent bacteria, preparation
of electrocompetent bacterial cells, transformation of electrocompetent bacteria. In
transformation of electrocompetent bacteria, plasmid DNA bacterial suspension and
transfer this mixture into a pre-chilled, sterile 0.2 cm gap cuvette. The salt
concentration in the DNA sample must be low. Insert the cuvette into the
electroporation chamber of the pulse control modul, and electroporate
and resuspending into LB broth for growth. Plate the bacteria onto LB agar plates
with appropriate selective agent.
3. Starter culture can out-compete a contaminant if there is one. That is more easily
accomplished with a starter culture, which is then used to inoculate a larger culture for
scale-up. Preparing competent cells will be easily reproducible.

Ref:
Gonzales MF, Brooks T, Pukatzki SU, Provenzano D. Rapid protocol for preparation
of electrocompetent Escherichia coli and Vibrio cholerae. J Vis Exp. 2013;(80):50684.
Published 2013 Oct 8. doi:10.3791/50684