QuikChange site directed mutagenesis method


Two-Stage PCR Protocol Allowing Introduction of Multiple Mutations, Deletions and Insertions Using QuikChange Site-Directed Mutagenesis

Biotechniques 26:680-682 (April 1999)

When longer primers are attempted (multiple and cassette mutagenesis, insertions and deletions), the mutagenesis efficiency is drastically decreased due to the more favorable primer dimer formation (100% complementary) compared to the primer-template annealing (due to multiple mismatches). The procedure consists of two stages. In stage one, two extension reactions are performed in separate tubes; one containing the forward primer and the other the containing the reverse primer.1-10 cycles are run. Subsequently, the two reactions are mixed, and the standard QCM procedure is carried out on the mixture.

Hosted by www.Geocities.ws

1