Abstract
The bacterial chromosome and bacterial plasmids can be engineered in vivo by homologous recombination using PCR products and synthetic oligonucleotides as substrates. This is possible because bacteriophage-encoded recombination proteins efficiently recombine sequences with homologies as short as 35 to 50 bases. Recombineering allows DNA sequences to be inserted or deleted without regard to location of restriction sites. This unit first describes preparation of electrocompetent cells expressing the recombineering functions and their transformation with dsDNA or ssDNA. It then presents support protocols that describe several two-step selection/counter-selection methods of making genetic alterations without leaving any unwanted changes in the targeted DNA, and a method for retrieving onto a plasmid a genetic marker (cloning by retrieval) from the Escherichia coli chromosome or a co-electroporated DNA fragment. Additional protocols describe methods to screen for unselected mutations, removal of the defective prophage from recombineering strains, and other useful techniques.
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