mutagenic primers with very high GC content.
Posted 25 June 2013 - 06:11 AM
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
Posted 25 June 2013 - 11:24 AM
Posted 30 June 2013 - 10:23 PM