Superman, on Oct 24 2009, 05:21 PM, said:
How can you work out the highest annealing temp possible? I read that annealing should be between 3-5 degrees lower than the Tm of the primers. Does anyone know if this is true. Sucks if it is cos I have a PCR running right now with annealing at 63 degrees and primer Tm's are around 55 degrees.
I am having a PCR nightmare atm!
I think you can do 10 different calculations and come up with 10 different answers. Annealing will depend on a number of factors including salt concentration, and different enzymes have different salt concentrations in the buffer. I use Pfu UltraII frequently, and it recommends either 3 or 5 degrees below the melting temp. Phusion, on the other hand, recommends 3 degrees over the melting temp because of a higher salt concentration.
Bottom line is that both of these are generally guidelines, but not set rules. It's a place to begin, and optimize from there. I've often used annealing temps that were higher than the given melting temp. You use what works.
For calculating melting temps, I typically use IDT's Oligo Analyzer ( http://www.idtdna.co.../OligoAnalyzer/
), but if you notice on that page, there's a place to put in different concentrations for different PCR components... if you change those values, the annealing temps change, so as I said, the melting temp is a starting point, not an absolute.