Enter the melting temperature (Tm) of the primer (commonly the lower Tm of the primer pair) and the melting temperature (Tm) of the PCR product (amplicon) to estimate the optimal annealing temperature for a PCR reaction.

Annealing Temperature Calculator

Enter any 2 values to calculate the missing variable

Annealing Temperature Formula

The following formula is used to estimate the optimal annealing temperature for a PCR reaction.

AT = (.3 * Tm primer ) + ( .7 * Tm product) - 14.9
  • Where AT is the estimated optimal annealing temperature (in °C when using this formula)
  • Tm primer is the melting temperature (Tm) of the primer (often the lower Tm of the primer pair)
  • Tm product is the melting temperature (Tm) of the double-stranded PCR product (amplicon)

Annealing Temperature Definition

An annealing temperature (Ta) is the temperature during the PCR annealing step at which primers hybridize (anneal) to the complementary template DNA sequence.

Example

Let’s take a look at an example of how to calculate the annealing temperature.

  1. First, we must determine the melting temperature of the primer. For this example, we will assume the temperature is 50 °C.
  2. Next, we must determine the melting temperature of the PCR product (amplicon). For this example, we will assume that it is 75 °C.
  3. Finally, we can calculate the annealing temperature using the formula above, which yields an estimated optimal annealing temperature of 52.60 °C.

FAQ

What is an annealing temperature?

An annealing temperature is the PCR step temperature at which primers anneal (hybridize) to the complementary template DNA sequence.