Hi, the annealing temperature should be 54-56 °C (you can do a gradient PCR). Cheers, Nadine

Hi Rene

You may do the touchdown pcr. You will get your required results definetely.

Best Regards

I would average it so it is 59 degrees, and Annelaing tem=Tm-5deg; so I would try from 54degrees.

I would go for gradient PCR from 52-58 with a increment of 1degree.

The most primers I use have a Tm 57-60. The annealing oC that I use is 59oC. For every primer pair I perform gradient PCR (50-70 oC) with different concentrations of MgCl2 (1.5-2.5mM). In this temperature (59) I observed the better results. Of course, it doesn't mean that they don't work in lower temperatures, like 54-56. However in order to be able to run many primer pairs, I use 59oC.

You must never forget that the tm you have calculate may be a little different than the real, as each software is using different parameters.