One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution.
We have developed a simple, one-step procedure for the preparation of competent Escherichia coli that uses a transformation and storage solution [TSS; 1 x TSS is LB broth containing 10% (wt/vol) polyethylene glycol, 5% (vol/vol) dimethyl sulfoxide, and 50 mM Mg2+ at pH 6.5]. Cells are mixed with an equal volume of ice-cold 2 x TSS and are immediately ready for use. Genetic transformation is equally simple: plasmid DNA is added and the cells are incubated for 5-60 min at 4 degrees C. A heat pulse is not necessary and the incubation time at 4 degrees C is not crucial, so there are no critical timing steps in the transformation procedure. Transformed bacteria are grown and selected by standard methods. Thus, this procedure eliminates the centrifugation, washing, and long-term incubation steps of current methods. Although cells taken early in the growth cycle (OD600 0.3-0.4) yield the highest transformation efficiencies (10(7)-10(8) transformants per micrograms of plasmid DNA), cells harvested at other stages in the growth cycle (including stationary phase) are capable of undergoing transformation (10(5)-10(7) transformants per micrograms of DNA). For long-term storage of competent cells, bacteria can be frozen in TSS without addition of other components. Our procedure represents a simple and convenient method for the preparation, transformation, and storage of competent bacterial cells.
Chung
C T
CT
Hepatitis Viruses Section, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892.
Niemela
S L
SL
Miller
R H
RH
D002413:Cations, Divalent; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods; D008274:Magnesium / Q000494:pharmacology; D010957:Plasmids; D013045:Species Specificity; D014169:Transformation, Bacterial / Q000187:drug effects
D002413:Cations, Divalent; D008274:Magnesium; D004121:Dimethyl Sulfoxide