Table 1.

Rules for high efficiency in vivo site-directed mutagenesis by Recombineering

Rule	Advice
One	Use dividing cells to enrich for replication intermediates.
Two	Use oligos complementary to the lagging strand template.
Three	Use oligos that incorporate a C•C mismatch in the paired recombination intermediate. Alternatively, use a 2+ nucleotide mismatch or knock out MMR.
Four	Plate transformants within 30–60 minutes of outgrowth.