Primer Tm differences/ PCR - (Dec/06/2010 )
I am using PCR primers from a publication. Although, forward and reverse primers have different Tms( 54C vs 48C or 42C vs 52C). Could anyone suggest what annealing tempetatures should I use? For the same primers the publication has an annealing temperatures of 47C and 50C. Any suggestions please?
I did use a pair of primers from that paper that had Tm 51 and 54C (good Tms as they are close), and I used annealing temperature of 47C which gave me great results.
Thank you very much!!
If you have access to a gradient PCR machine, or know of someone that does, I would strongly recommend running a gradient PCR program that spans the annealing temperatures you describe. This is the best way to determine what annealing temperature works best for primers with very different annealing temperatures.
If you do not have access to a gradient PCR machine, then your best bet would be to start with the lower annealing temperature. This is because if you use the higher annealing temperature then much less of the primer with the lower annealing temperature will bind to the DNA, thus leading to smaller amounts of DNA amplification. You may get additional non-specific amplification/background, but that is something you may to have to make due as a results of primer design.