How do I calculate the melting temperature (Tm) of primers?
How do I calculate the melting temperature (Tm) of primers?
The primer melting temperature (Tm) is the estimate of DNA-DNA hybrid stability. Knowing the Tm is critical for determining an appropriate annealing temperature (Ta). A Ta that is too high will result in insufficient primer-template hybridization, leading to low PCR product yield. A Ta that is too low may lead to non-specific product amplification. Calculation of the Tm of primers shorter than 20 bases can be performed using the Wallace rule: Tm = 2°C (A+T) + 4°C (G+C) For accurate estimation of the Tm of primers longer than 20 bases, we recommend using free primer design software such as Primer3.