title: Improved method for high-efficiency electrotransformation of Escherichia coli with the large BAC plasmids.
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 7 × 10(8) 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.
authors.affiliation: Department of Microbiology, Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, 84551, Bratislava, Slovakia.
authors.forename: Jana
authors.initials: J
authors.lastname: Nováková
authors.forename: Anita
authors.initials: A
authors.lastname: Izsáková
authors.forename: Tomáš
authors.initials: T
authors.lastname: Grivalský
authors.forename: Christian
authors.initials: C
authors.lastname: Ottmann
authors.forename: Marian
authors.initials: M
authors.lastname: Farkašovský
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods; D022202:Chromosomes, Artificial, Bacterial; D018274:Electroporation / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D018014:Gene Transfer Techniques; D005827:Genetics, Microbial / Q000379:methods*; D010957:Plasmids; D014169:Transformation, Bacterial