Combined Overlap Extension PCR Method for Improved Site Directed Mutagenesis

Figure 4

Results of COE-PCR for the 6th primer set. The first primary PCR reaction required optimization before producing the desired band in sufficient quantity. Before optimization (lane 1), the PCR reaction produced a faint band estimated at 1.4 kb, which matched the expected size. After optimization (lane 3), PCR produced multiple bands, with a bright band estimated at 1.4 kb (indicated by white arrow), which matched the expected size. Thus, the 1.4 kb band was excised and purified. The second primary PCR reaction (lane 2) produced a single band estimated at 500 bp (indicated by white arrow), which matched the expected size. The secondary PCR reaction (lane 4) produced a bright band estimated at 1.9 kb (indicated by black arrow), which matched the expected size.