PMID:21815084
| Citation |
Murphy, KC (2011) Targeted chromosomal gene knockout using PCR fragments. Methods Mol. Biol. 765:27-42 |
|---|---|
| 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. |
| Links |
PubMed Online version:10.1007/978-1-61779-197-0_2 |
| Keywords |
Chromosomes, Bacterial/genetics; Escherichia coli/genetics; Gene Knockout Techniques/methods; Genes, Bacterial/genetics; Polymerase Chain Reaction |
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