Preparation of highly efficient electrocompetent Escherichia coli using glycerol/mannitol density step centrifugation.

Traditional protocols for preparing Escherichia coli for electroporation are laborious and often deliver highly variable transformation efficiencies. Many laboratories resort to purchasing expensive commercially prepared cells. This article describes a simple method for producing electrocompetent E. coli by centrifuging bacteria through a glycerol/mannitol density cushion. The method is rapid and replaces tedious multistep procedures with two 15-min centrifugations. Standard cloning strains consistently produce more than 8×10(9)transformants/μg pUC18, whereas the strains TG1 and LE392 display efficiencies of more than 3×10(10)/μg DNA.