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problems with mutant PCR - problems with mutant PCR (Jul/26/2007 )


I am trying to amplify a 4.7 kb plasmid for site directed mutagenesis using PfuUltra from Stratagene. I designed the complementary mutant primers with the desired mutation right in the middle ( A to G substitution) using the primerX program recommended by Stratagene. The size of the primers are 28 bp and their Tm is between 76-79 degrees C. The oligoanalysis theoretically predicts that there are no hairpins or dimers that are formed. I get absolutely no product when I do the PCR at different annealing temperatures ranging from 55-70 degrees. A positive control that I have run for every PCR with a known set of primers and template has always worked. I have also checked the template to ensure it is not degraded and have also confirmed it by restriction digestion to see if it is the correct template.
Specific queries:
1. Does addition of DMSO improve the results? Does the DMSO have to be a recent batch or can I use any DMSO?
2. Can a mixture of Taq polymerase and PfuUltra alleviate the problem? (I have read some previous literature pertaining to this)
3. Does varying the template concentration help to solve the problem? What is the recommended concentration? I have used 10-40ng/microlitre.
4. Is there an alternative kit or enzyme system I can use such as a Long template PCR to solve the problem. Using Pfuultra has been very problematic for this particular PCR.
5. Are there any specific precautions one should take when doing PCR for site directed mutagenesis? Should the mutation be right in the centre always?





I don't know the answers to all of your specific questions. However, one trick that I've found helpful is to increase the number of PCR cycles I do. I believe the Stratagene kit recommends 12 cycles, but I've found that 18 cycles tends to work better for me. And I use 5-50 ng of template in the total reaction. See for details.

Also, you may want to check your plasmid of a AT rich region. Sometimes, such regions can cause problems in long PCRs.

Good luck.