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PCR Annealing Temperature Calculator — Primer Pair, ΔTm & Gradient Range
Estimate a practical PCR annealing-temperature starting point from forward and reverse primer Tm values, flag mismatched primer pairs and generate a gradient range for empirical optimization.
Last updated: 2026-09-17
Results
Enter both primer Tm values above 0 °C
Formula, example and assumptions
Starting Ta≈lower primer Tm−5 °C; gradient shown around the starting estimate.
For primers at 60 °C and 61 °C Tm, a 55 °C starting annealing estimate with a nearby gradient is a useful first experiment.
- Annealing temperature is protocol- and polymerase-dependent; manufacturer guidance takes priority.
- The result is a starting point for empirical optimization, not a guarantee of amplification specificity or yield.
Frequently asked questions
Why use the lower primer Tm?
A shared annealing step must allow both primers to bind; a simple starting rule therefore anchors to the less thermally stable primer.
What if primer Tm values differ a lot?
A large ΔTm is a design warning. Rather than forcing a compromise temperature, consider redesigning the pair and verify it experimentally.