Fig 1. Unparalleled mutagenesis rate achieved by TagMaster^® Site-Directed Mutagenesis Kit.

Mutagenic primers were designed to substitute 2bp or insert a 6His tag into the LacZ gene in pUC19. The mutagenic primer (gtacccggggatcctctCAagtcgacctgcaggcat) and its reverse complement primer were used to substitute 2bp in the LacZ gene. The mutagenic primer (gcctgcaggtcgactCACCACCACCACCACCACtagaggatccccggg) and its reverse complement primer were used to insert a 6His tag into the LacZ gene. pUC19 plasmid purified from DH5α was used as template. Three mutagenesis reactions were performed by using TagMaster Enzymes and 100ng of pUC19. The reaction conditions were 95°C for 5min, followed by 22 cycles of 95°C 20sec, 60°C 10sec, 68°C 2min. Mutagenic primers were not added in one reaction (No primer Control), as a negative control. Without any treatment, the products of reactions were directly transformed into TagMaster competent cells and spread onto LB plates. The clone number in each plate was counted as shown in panel A. Only 5 clones grew in the control plate, which demonstrated that TagMaster cells efficiently eliminated the template plasmid DNA. In contrast, hundreds of clones grew in the other two mutagenesis groups, which showed TagMaster cell's ability to specifically enrich newly synthesized mutagenic DNA. The mutagenesis rates were calculated by the formula: (clone counts - control clone counts) / clone counts, and results are shown in panel B. All 4 primers used here are provided in the kit as control primers. Please see the kit's manual for the detailed procedure to design mutagenic primers.