Transformation of Escherichia coli increases 260-fold upon inactivation of T4 DNA ligase.
It was possible to obtain high-efficiency transformation of E. coli MC1061 by the following modifications of the standard procedure: cells were harvested at A600 of 550-650, washed with 1, 1/2, and 1/40, and were resuspended in 1/500 culture vol of 1 mM Hepes, pH 7.0, to a cell concentration of 6 x 10(10)-6 x 10(11) cells/ml. Electrocompetent cells were used immediately for electroporation to yield 1.3 +/- 0.5 x 10(9) (mean +/- SD) transformants micrograms of plasmid DNA, which is comparable to the efficiency of bacteriophage lambda infection. Alternatively, cells can be stored frozen in 10% glycerol, although glycerol reduced transformation efficiency to approximately 30% (data not shown). Freezing and thawing of glycerol-treated cells did not result in any further loss of transformation efficiency (data not shown). This study showed that it is crucial to inactivate the T4 DNA ligase prior to electrotransformation of ligated DNA, which can be ensured by the introduction of a simple heat inactivation step, increasing the number of transformants by 260-fold. Although this paper focuses on the use of E. coli MC1061/p3, the experiments were repeated with a different plasmid in the parental strain E. coli MC1061 and showed the same result (data not shown.(ABSTRACT TRUNCATED AT 250 WORDS)
Michelsen
B K
BK
Hagedorn Research Institute, Gentofte, Denmark.
D001431:Bacteriological Techniques; D017122:Bacteriophage T4 / Q000201:enzymology*; D011088:DNA Ligases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004558:Electric Stimulation / Q000379:methods; D004926:Escherichia coli / Q000235:genetics*; D005821:Genetic Techniques; D014169:Transformation, Bacterial
D011088:DNA Ligases