Sauer:Plasmid-born λRed recombineering using dsDNA
This protocol was taken (with minor modifications) from Thomason et. al. Current Protocols in Molecular Biology (2007) 1.16.1-1.16.24
Using short (35-40 bp) regions of homology, bacteriophage-encoded recombination machinery allows for the efficient insertion or deletions in gram-negative bacterial chromosomes without regard to restriction sites. The Court lab has extended the "Wanner" method by placing the plasmid-born bacteriophage recombination functions under the control of a temperature sensitive allele lambda repressor (cI857). At low temperatures (30-34 C) the downstream genes are tightly repressed however following a temperature shift to 42 C, these genes are expressed at high levels from the lambda pL promoter. Following induction of the recombination functions, transformed dsDNA is efficiently recombined into the chromosome and recombinants identified by either selection or PCR based screening. Some of the Court lab plasmids carry a temperature sensitive origin allowing for facile plasmid curing by simply shifting to the non-permissive temperature (37) and omitting the selection marker (chloramphenicol in the case of pSIM5).