Calculate PCR annealing temperature, primer melting temperature, or amplicon melting temperature from any two values in °C or °F.

Annealing Temperature Calculator

Enter any 2 values to calculate the missing variable

Annealing Temperature Formula

The annealing temperature calculator uses the PCR annealing temperature equation that combines the primer melting temperature and the PCR product, or amplicon, melting temperature. The constant 14.9 is based on temperatures in degrees Celsius, so Fahrenheit inputs are converted to Celsius before the formula is applied.

Tₐ = 0.3*Tₘ(primer) + 0.7*Tₘ(product) - 14.9

To calculate the primer melting temperature instead:

Tₘ(primer) = (Tₐ - 0.7*Tₘ(product) + 14.9) / 0.3

To calculate the PCR product melting temperature instead:

Tₘ(product) = (Tₐ - 0.3*Tₘ(primer) + 14.9) / 0.7
  • Ta = annealing temperature
  • Tm(primer) = melting temperature of the primer
  • Tm(product) = melting temperature of the PCR product, also called the amplicon
  • 14.9 = empirical temperature adjustment used by this equation, in degrees Celsius

If you enter the primer melting temperature and the PCR product melting temperature, the calculator finds the annealing temperature. If you enter the annealing temperature and one melting temperature, it rearranges the same equation to solve for the missing melting temperature. Inputs can be entered in °C or °F, but the calculation is performed in °C and then converted back to the selected output unit.

Typical PCR Temperature Ranges

These ranges are general reference values. Actual PCR conditions can vary by polymerase, buffer chemistry, primer design, target sequence, and cycling protocol.

PCR value Common range Notes
Primer Tm 50 to 65 °C Forward and reverse primers are usually designed with similar melting temperatures.
Annealing temperature 50 to 65 °C Often optimized by gradient PCR if nonspecific bands or weak amplification occur.
Amplicon Tm Depends on length and GC content Longer and GC-rich products usually have higher melting temperatures.

Annealing Temperature Examples

Example 1: Calculate annealing temperature

Suppose the primer melting temperature is 60 °C and the PCR product melting temperature is 75 °C.

Tₐ = 0.3*60 + 0.7*75 - 14.9
Tₐ = 18 + 52.5 - 14.9 = 55.6 °C

The annealing temperature is 55.60 °C.

Example 2: Calculate primer melting temperature

Suppose the annealing temperature is 58 °C and the PCR product melting temperature is 78 °C.

Tₘ(primer) = (58 - 0.7*78 + 14.9) / 0.3
Tₘ(primer) = (58 - 54.6 + 14.9) / 0.3 = 61.0 °C

The primer melting temperature is 61.00 °C.

Annealing Temperature FAQ

What is annealing temperature in PCR?

Annealing temperature is the temperature during PCR where primers bind to the template DNA. If the temperature is too low, primers may bind nonspecifically. If it is too high, primers may not bind efficiently, which can reduce or prevent amplification.

Should the annealing temperature be lower than the primer Tm?

In many PCR setups, the annealing temperature is set a few degrees below the primer melting temperature. This calculator uses a formula that also includes the PCR product melting temperature, so the result may not equal a simple fixed offset below primer Tm.

What should you do if the calculated annealing temperature does not work well?

Use the calculated value as a starting point. If amplification is weak, you may test slightly lower annealing temperatures. If nonspecific products appear, you may test slightly higher annealing temperatures. A gradient PCR is a common way to test several annealing temperatures in one run.