Why subtract from the lower Tm, not the average?

Both primers must bind for exponential amplification. Anchoring on the lower-Tm primer ensures it anneals; using the average can leave the weaker primer poorly bound.

What if my primers have very different Tm values?

A gap over ~5 °C makes a single annealing temperature a compromise. Redesign so the Tm values match, or use a touchdown protocol.

PCR annealing temperature calculator

Set a PCR annealing temperature from your two primer melting temperatures — the common rule of thumb is about 5 °C below the lower primer Tm.

How it works

Formula

annealing temperature Ta = min(primer Tm₁, primer Tm₂) − offset. The offset is typically about 5 °C. Use the lower of the two primer Tm values so both primers anneal.

Worked example

Primers with Tm 58 °C and 60 °C, offset 5 °C: 58 − 5 = 53 °C annealing temperature.

When to use it

As a starting annealing temperature when setting up a new PCR. If you see non-specific bands, raise Ta; if the product is weak or absent, lower it. Keep the two primer Tm values within ~5 °C of each other.

Sensible defaults

Defaults use a 58/60 °C primer pair and a 5 °C offset → 53 °C. Feed in Tm values from the nearest-neighbor Tm calculator for the best estimate.

FAQ

Why subtract from the lower Tm, not the average?: Both primers must bind for exponential amplification. Anchoring on the lower-Tm primer ensures it anneals; using the average can leave the weaker primer poorly bound.
What if my primers have very different Tm values?: A gap over ~5 °C makes a single annealing temperature a compromise. Redesign so the Tm values match, or use a touchdown protocol.