Combined Overlap Extension PCR Method for Improved Site Directed Mutagenesis

Figure 3

Results of COE-PCR for the 4th primer set. Both primary PCR reactions (lanes 1 and 2) produced single bands estimated at 1050 bp and 900 bp (indicated by white arrows), which matched the expected size and did not require gel purification. Lane 3 shows the results of the secondary PCR reaction after a single cycle, indicating that a single cycle was insufficient to amplify sufficient DNA. The secondary PCR reaction (lane 4) produced a bright band estimated at 1.9 kb (indicated by black arrow), which matched the expected size, and two additional bands of higher molecular weight. Since the 1.9 kb band matched the expected size, it was carefully excised, purified, and used as the template for the next mutagenesis experiment.