Anyone with experiences in 2 step PCR - (May/07/2009 )
Hi, the PCR I'm currently optimising is producing multiple bands. I've got it down to two bands but I'm currently annealing at 60oC. I thought about touchdown PCR or just going high with the annealing temp, but once I go over the magic 60oC mark I have the probelm that my Taq starts extension at 62oC. I've heard of 2 step PCR where you can run 1) denaturation and 2) a combined anealing/ extension temp between 60-70oC. Has anyone used this method before? Does it work?
Any other suggestions would be helpful too.
Works fine, many of the real-time PCR runs I do are two step.