1. DNA DNA template plasmid 5-20 ng 2. PCR conditions 95°C 30 seconds, 3. Degradation of methylated (parental) DNA with DpnI Cool down PCR reaction. 4. Transformation into E. coli Place 200 µl highly competent cells (1 x 108/ug efficiency or greater) DH5 a on ice. 5. Miniprep 6 colonies and digest plasmids looking for mutant. 6. Sequence the lesion to confirm the change. Notes: 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 lesions Note that this is a linear amplification, so that the template only gets copied one time. Hence the odds of introducing lesions when using a high fidelity polymerase are very low. Analysis of PCR product by gel electrophoresis Run an agarose gel ( 5 -10 µl). If the product is visible, the mutagenesis will almost certainly work. Even if the product is not visible, one often get the mutant. 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 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. |
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