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Folia Microbiol (2014) 59:5361 DOI 10.1007/s12223-013-0267-1
Improved method for high-efficiency electrotransformation of Escherichia coli with the large BAC plasmids
Jana Novkov & Anita Izskov & Tom Grivalsk &
Christian Ottmann & Marian Farkaovsk
Received: 16 January 2013 /Accepted: 28 June 2013 /Published online: 12 July 2013 # Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2013
Abstract High transformation competency of Escherichia coli is one of the critical factors in the bacterial artificial chromosome (BAC)-based DNA library construction. Many electroporation protocols have been published until now, but the majority of them was optimized for transformation of small plasmids. Large plasmids with a size above 50 kbp display reduced transformation efficiency and thereby require specific conditions in the preparation and electroporation of electrocompetent cells. In the present work, we have optimized the parameters critical to the application of BAC DNA electrotransformation into E. coli. Systematic evaluation of electroporation variables has revealed several key factors like temperature of growth, media supplements, washing buffer, and cell concentration. Improvements made in the transformation protocol have led to electrocompetent cells with transformation efficiency up to 7108 transformants per microgram of 120 kbp BAC plasmid DNA. We have successfully used in-house prepared competent cells, the quality of which is comparable with those produced by different companies, in the construction of metagenomic libraries from the soil. Our protocol can also be beneficial for other application with limited DNA source.
Introduction
Methods for transfer of exogenous DNA into cells play a crucial role in genetics and molecular biology. A number of
methods have been developed to artificially transfer DNA into cells. Two major techniques, chemotransformation and electroporation, have been used to introduce DNA into bacterial cells. Several other methods were also described, including transformation of protoplasts and spheroplasts, the freeze and thaw technique, biolistic transformation, sonoporation, liposome-mediated DNA transfer, chitosan-mediated transformation, and tribos transformation. However, most of them have only a narrow range of use, limited transformation efficiency, or require expensive equipment (Aune and Aachmann 2010). Historically oldest method, chemical transformation, uses treatment with polyvalent cations and heat shock for transient opening gated membrane channels. Since the first demonstration of chemical transformation method (Mandel and Higa 1970), many different procedures have been developed with variable transformation efficiencies. A detailed...