How should I determine the annealing temperature for my qPCR reaction? Should I go midway between my primer and probe Tms?
When doing RT-PCR it is ideal to have your probe Tm about 10 degrees higher than your primer Tms. Your annealing temperature should be set about 5 degrees lower than the lowest primer Tm. This is a general guide to follow, optimization may still be necessary, but this is a good starting point.