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[43] Preparation and storage of competent Escherichia coli cells

1993; Academic Press; Linguagem: Inglês

10.1016/0076-6879(93)18045-e

1557-7988

Cathie T. Chung, R.H. Miller,

Transgenic Plants and Applications

Publisher Summary The uptake of foreign DNA by Escherichia coli can be induced either through electroporation, which involves discharging an electrical voltage across bacterial cell membranes, or by making bacteria competent through chemical methods. Two widely used chemical methods involve treating bacteria with calcium chloride or hexamine cobalt and subjecting the cells to a heat shock. The protocol developed for the preparation of competent E. coli was designed to be rapid, convenient, and inexpensive, yet suitable to achieve transformation efficiencies sufficient for routine cloning experiments. Although transformation efficiencies obtained with the protocol are comparable to those of other commonly used methods to prepare competent bacteria, the technique is advantageous in that it is simple and can be completed in a short time once the bacteria are grown. Although the conditions necessary to achieve maximum numbers of transformants are specified, there is flexibility in the assay.