Datsenko (2000) demonstrated one-step inactivation of chromosomal genes using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated (H1 and H2) and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. But with some exceptions, the paper describing the Keio Collection (Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection BABA et al. 2006) used 50-nt homology in both H1 and H2 regions.
I want to use 40-nt extensions in H1 and H2 because the primer price is lower. Does anyone know of any restrictions associated to that? Does the primer need to be PAGE purified?
40 nucleotides primers should also work in principle. The primers should be pcr ready.
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