A rapid and highly efficient method for preparation of competent Escherichia coli cells.

This protocol describes a simple and efficient procedure for preparation and storage of competent Escherichia coli cells applicable to transformation and transfection. This procedure is much simpler and more reproducible than other similar methods, and allows much higher transformation efficiency (1) even after 3 months storage of the competent cells. It is also superior to the electroporation (2) in that it does not require any expensive apparatus, and gives comparable, but more consistent results. The procedure consists essentially in cell growth in medium containing Mg + and carbon source and storage in glycerinPEG (polyethylene glycol)-Mg at -80°C. Bacteria were made competent by the following procedures 50 ml culture inoculated with 0.5 ml of over-night culture was grown with aeration in medium A (LB broth supplemented with 10 mM-MgSO4 7H2O and 0.2%-glucose) to mid logarithmic phase. The presence of 10 mM-Mg in medium stimulates transformation efficiency (3). The increased growth rate due to extra carbon source (glucose here) also enhances transformation efficiency. The cells were kept on ice for 10 min, then pelleted at 1500 g for 10 min at 4°C. The cells were resuspended gently in 0.5 ml of medium A precooled on ice, then 2.5 ml of storage solution B (36%-glycerin, 12%-PEG (MW7500), 12 mMMgSO4 7H2O added to LB-broth (pH 7.0) and sterilized by filtration) was added, and mixed well without vortexing. The competent cells were divided in aliquots of 0.1 ml each in Eppendorf tubes and stored at —80°C until use. Rapid freezing in dry ice/ethanol is not needed; however, cells must be handled gently on ice during all processes before freezing. Transformation efficiency would decrease by 10~ if a culture was centrifuged and resuspended at room temperature.