Fidelity testing workflow

Polymerase Fidelity Testing at NEB is based on error accumulation in the lacZ gene during PCR. A technique developed by Wayne Barnes.

First, the lacZ gene is amplified by PCR with the polymerase of interest, leading to the accumulation of errors at the polymerase's intrinsic rate of error incorporation. The amplified DNA is digested by the restriction enzymes NcoI and HindiIII, and inserted into a plasmid while restoring two antibiotic resistance genes.

The plasmids are then transformed into competent E. coli. The E. coli are either grown on selective media containing X-gal, so that blue and white colonies can be counted, or grown on selective media, isolated, then sequenced.