Figure 4

PfuX7 polymerase is compatible with the Kunkel method for template elimination in site-directed mutagenesis. (A) Screen designed to calculate the efficiency of site-directed mutagenesis. By introducing a stop codon mutation in a plasmid responsible for constitutive expression of the green fluorescent protein in E. coli cells, the efficiency of the mutagenesis is easily calculated as the ratio of non-fluorescent cells to total amount of cells. Shown, is a typical example of an experiment using template DNA isolated from either DH5α or CJ236. (B) Agarose gel electrophoresis of PCRs performed with the PfuTurbo- (T) or the PfuX7 (×7) DNA polymerases using template plasmid DNA isolated from the ung+ E. coli strain DH5α or the ung- strain CJ236. The molecular marker (M) is kb+ (Invitrogen). (C) Quantification of the efficiency of site-directed mutagenesis using the PfuTurbo- or the PfuX7 DNA polymerases using template plasmid DNA isolated from the ung+ E. coli strain DH5α or the ung- strain CJ236, with or without DpnI treatment. Data represents the average of three independent experiments with standard deviations.