How accurate is the Wallace rule?

It is a rough rule of thumb that ignores salt concentration, primer concentration and sequence context. It is fine for picking between candidate short primers, not for exact annealing temperatures.

What annealing temperature should I run?

A common starting point is about 5 °C below the lower of your two primer Tm values, then optimise empirically.

Primer melting temperature (Wallace rule)

Estimate the melting temperature of a short DNA oligo with the Wallace "2 + 4" rule, plus GC content, length and reverse complement.

How it works

Formula

Tm = 2 °C × (A + T) + 4 °C × (G + C)

Worked example

For ACGTACGT there are 4 A/T bases and 4 G/C bases: (2 × 4) + (4 × 4) = 8 + 16 = 24 °C.

When to use it

A quick desk estimate for short primers (under roughly 14 nt). For longer oligos or precise PCR design, use a nearest-neighbour model that accounts for salt and primer concentration.

Sensible defaults

The default ACGTACGT is a balanced 8-mer that lands at 24 °C — handy for sanity-checking the rule.

FAQ

How accurate is the Wallace rule?: It is a rough rule of thumb that ignores salt concentration, primer concentration and sequence context. It is fine for picking between candidate short primers, not for exact annealing temperatures.
What annealing temperature should I run?: A common starting point is about 5 °C below the lower of your two primer Tm values, then optimise empirically.