| Mutagenic primers were designed to substitute 2bp or insert a 6His tag into LacZ gene in pUC19. The mutagenic primer
(gtacccggggatcctctCAagtcgacctgcaggcat) and its reverse complement primer were used to substitute 2bp in Lac Z gene. The mutagenic primer
(gcctgcaggtcgactCACCACCACCACCACCACtagaggatccccggg) and its reverse complement primer were used to insert a 6His tag into Lac Z gene. pUC19 plasmid purified from
DH5¦Á was used as template. Three mutagenesis reactions were performed by
using TagMaster Enzymes and 100ng of pUC19. The reaction
conditions were 95¡ãC for 5min, followed by 22 cycles of
95¡ãC 20sec, 60¡ãC 10sec, 68¡ãC 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. Clone number in each plate were counted as shown in panel A.
Only 5 clones grew in the control plate, which
demonstrated that TagMaster cells efficiently eliminated 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 the 4 primers used here are provided in the kit as control primers. Please see the kit's manual for the detail procedure to design mutagenic primers. |
