*Also read manual from Stratagene http://www.stratagene.com/manuals/200518.pdf

DpnI mediated site-directed Mutagenesis

A highly effective simple method for making specific mutations in plasmids without subcloning: based on the work of Fisher and Pei (1997).

1. Amplification of mutant DNA

PCR conditions

2. Degradation of methylated (parental) DNA with Dpn I

3. Transformation into E. coli (see separate protocol for super-competent cells)

4. Miniprep 6 colonies

5. Sequence the clones to confirm the change


The Stratagene QuickchangeTM site-directed mutagenesis protocol provides a detailed description of the approach. The manual is available as a PDF file at http://www.stratagene.com/manuals/200518.pdf

Design of oligos

Plasmid size

Introduction of mutations

Using other polymerases


Fisher, C. L., and Pei, G. K. (1997). Modification of a PCR-based site-directed mutagenesis method. Biotechniques 23, 570-574.