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Targeted Chromosomal Gene Knockout Using PCR Fragments

  • Kenan C. MurphyEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 765)

Abstract

The development of recombineering technology has converged to a point that virtually any type of genetic modification can be made in the Escherichia coli chromosome. The most straightforward ­modification is a chromosomal gene knockout, which is done by electroporation of a PCR fragment that contains a selectable drug marker flanked by 50 bp of target DNA. The phage λ Red recombination system expressed in vivo from a plasmid promotes deletion of the gene of interest at high efficiency. The combination of this technology with site-specific recombination systems of Cre and Flp has enabled genetic engineers to construct a variety of marked and precise gene knockouts in a variety of microbial chromosomes. The basic protocols for designing PCR substrates for recombineering, generating ­recombineering-proficient electrocompetent strains of E. coli, and for selection and verification of recombinant clones are described.

Key words

Recombineering Lambda red Gene replacement Strain development Electroporation Phage lambda Beta Exo Gam PCR 

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Microbial and Physiological systemsUniversity of Massachusetts Medical SchoolWorcesterUSA

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