mutagenic primers with very high GC content. - (Jun/25/2013 )
I am trying to introduce a few point mutations, using an inverse PCR method (analogous to Stratagene QuikChange Sitedirected Mutagenesis). I have got my mutagenic primers synthesized from Macrogen Inc, and carried out the reaction manually (did not use any kit). I did manage to get a few positive clones, however a couple of mutants just would not come.
Interestingly for these two cases, the primers are of very high GC content (>70%). I have tried with 1% DMSO in the reaction mix, however, it still fails.
Moreover, when i did see colonies, they turn out to be false positives. I am using NEB DpnI for digesting the parent plasmid post PCR amplification, and it seems to be working fine.
Can anyone suggest where the problem lies?
I will be happy to provide any other information regarding the reaction mix if needed.
Note: I use desalted primers, and carry out PCR cleanup prior to DpnI digestion
You could try 2-5% of a 1M betaine solution, which in my experience works wonders for high GC reactions. Be sure to use a non-strand displacing enzyme.
Thanks a lot. I will try out the suggested modifications. I have been using Phusion Polymerase for my reactions, and notable I did manage to get a few mutants done. Can I still continue with it or do I need to find an alternative?
Phusion should be fine.