Annealing Temperature Calculator

Use this free online Annealing Temperature Calculator to estimate the optimal Tm (melting temperature) for your PCR primers. This calculator uses the Wallace Rule and is ideal for both beginner and professional-level molecular biologists.

Formula Used (Wallace Rule): Tm = 2°C × (A+T) + 4°C × (G+C)

Number of A: Number of T: Number of G: Number of C: Calculate Tm

What is Annealing Temperature in PCR?

The annealing temperature is the temperature at which primers bind to the DNA template during PCR. Choosing the correct Tm helps prevent non-specific binding and increases amplification accuracy.

How to Calculate Tm for PCR?

The Wallace Rule is widely used for short DNA primers: Tm = 2°C × (A+T) + 4°C × (G+C). Other advanced formulas like Nearest Neighbor Method may be used for longer or complex sequences.