Protocol

Rapid PCR Site-Directed Mutagenesis

This protocol was adapted from "Rapid PCR Site-Directed Mutagenesis," contributed by Michael P. Weiner and Gina L. Costa, Chapter 31, in PCR Primer, 2nd edition (eds. Dieffenbach and Dveksler). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.

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INTRODUCTION

This PCR method of site-directed mutagenesis (SDM) has many advantages, which include the following:

Use of increased template concentration, which allows fewer cycles to be used, thus reducing the potential for amplifying nonspecific products; inclusion of Taq Extender, which provides increased yield and reliability in the amplification of longer PCR products; use of DpnI restriction endonuclease, which reduces the number of parental molecules; use of Pfu DNA polymerase to remove undesired base extensions, which improves the efficiency of blunt-end ligation; and the complete protocol can be performed rapidly and does not require purification or precipitation procedures between steps. PCR-SDM . . . [Full Text of this Article]

MATERIALS

Reagents

Equipment

METHOD