How should I determine the annealing temperature for a probe-based qPCR assay? Should I select a Tm midway between my primer and probe Tms?
When performing qPCR it is ideal to have your probe Tm about 5-10 degrees higher than your primer Tms. The annealing temperature should be set 3-5 degrees lower than the lowest primer Tm. Use this as a general guideline, but note that optimization may still be necessary.