*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

• DNA template plasmid 5-20 ng
• 10x Pfu DNA polymerase buffer 5.0 µl
• 25uM forward primer 0.5 µl
• 25uM reverse primer 0.5 µl
• 25mM dNTP 1.0 µl
• Pfu DNA polymerase (2.5 units) 1.0 µl
• fill w/ddH2O to 50 µl

PCR conditions

• 95° 30 seconds,
• 18 cycles of : 95° 30 seconds, 55° 1 minute, 68° 2 minutes/kb of plasmid length

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

• Cool down PCR reaction.
• Add 1µl Dpn I (10 units) to PCR reaction 37°C and incubate for 1 hour
• Can run DNA gel to confirm

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

4. Miniprep 6 colonies

5. Sequence the clones to confirm the change

Notes:

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

• Oligos should be perfectly complementary (actually haven't checked out if this in required). The oligos should contain 12-15 bp on each site of the lesion. It is useful to engineer a restriction site addition or loss into the oligos for ease of screening. We have used the protocol for deletions using 17 bp on each side of the deletion.

Plasmid size

• We have used the protocol for plasmids over 18 kb in length. Plasmids below 10 kb seem to work routinely. In most cases, a vast majority 80-100% of colonies are correct. For longer plasmids it may be necessary to work with PCR to optimize synthesis.

Introduction of mutations

• Note that this is a linear amplification, so that the template only gets copied one time. Hence the odds of introducing unwanted mutations when using a high fidelity polymerase are very low.

Using other polymerases

• We have started using TaKaRa Ex Taq instead of Pfu polymersase with great success using large plasmids (15-20 kb size). While getting the amplification to work efficiently with Pfu can take a bit of fiddling, amplification with Ex Taq appears very robust for large plasmids.

REFERENCES

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