Biology

Annealing Temperature Calculator

Estimate PCR annealing temperature from primer melting points.

Calculated Output

Annealing temperature (°C) 55

Formula: Ta = min(Tm₁, Tm₂) − offset

Step-by-Step Breakdown

1. Values used:

2. Primer 1 Tm = 60 °C

3. Primer 2 Tm = 62 °C

4. Offset below lowest Tm = 5 °C

6. Annealing temperature = 55°C

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The math behind it

A common rule of thumb: set the annealing temperature ~5 °C below the lower of the two primer melting temperatures.

Tm 60 & 62 → 60 − 5 = 55 °C.

Optimize further?

Run a temperature gradient around this value for the cleanest product.