Figure 2

Agarose gel representing the results of megaprimer based PCR mutagenesis products and effect of limiting first flanking primer concentration on mutation frequency. Lanes 1, 2 and 3 are results obtained using 0.05, 0.1 and 1.0 pmole of the first flanking primer (T7 terminator) in the first PCR reaction. Lane M contains 1 μg of GeneRuler™ DNA ladder mix. The second PCR (1837 bp) product was purified from the gel. It was subjected to double restriction digestion using BamHI &HindIII. The resulting fragment (1482 bp) was cloned into the expression vector. The clones were sequenced to calculate the mutagenesis frequency.