title: Preparation of highly efficient electrocompetent Escherichia coli using glycerol/mannitol density step centrifugation.
abstract: 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.
authors.affiliation: Department of Medical Biochemistry at Radiumhospitalet, Oslo University Hospital, Montebello, N-0310 Oslo, Norway. david.warren@radiumhospitalet.no
authors.forename: David J
authors.initials: DJ
authors.lastname: Warren
mesh_terms: D002499:Centrifugation, Density Gradient / Q000379:methods; D018274:Electroporation / Q000379:methods*; D004926:Escherichia coli / Q000166:cytology* / Q000302:isolation & purification; D005990:Glycerol / Q000737:chemistry*; D008353:Mannitol / Q000737:chemistry*
chemical_list: D008353:Mannitol; D005990:Glycerol