primer Tm for PCR mutagenesis - (Nov/29/2007 )
I'm trying to design primers for a PCR mutagenesis (I want to delete 20 bases). What should the Tm of my primers be? Some sites say 55- 68c is good otheres sites say 55-72 is good. And i've found that tm calculations vary alot depending on which website I use which confuses me even more.
20 bases is a big deletion. Because the gap is so large, you need to treat each half of your primer/s as though they are separate primers. I always think the higher the Tm the better because the higher annealing temperature, the higher the specificity, so 72C for me - that's 72C on each half of each primer. Once you have the two PCR products they'll contain the desired overlap and you can PCR them together.