PCR Primer Annealing Temperature Calculator
Annealing temperature in PCR - what this calculator does
This calculator estimates a practical PCR annealing temperature using your forward and reverse primer sequences. It first estimates each primer’s melting temperature (Tm) and then recommends an annealing temperature for the primer pair.
How primer Tm is estimated here
For simplicity and broad usability, this calculator uses the classic Wallace rule, which works best as a quick estimate for typical primer lengths.
Wallace rule:
\[ Tm \approx 2 \cdot (A + T) + 4 \cdot (G + C) \]
Where \(A\), \(T\), \(G\), and \(C\) are the counts of each nucleotide in the primer sequence.
From Tm to annealing temperature
A common practical guideline is to set annealing temperature a few degrees below the lower primer Tm:
\[ Ta \approx \min(Tm_{F}, Tm_{R}) - 3 \]
Because real PCR conditions vary (buffer, MgCl2, additives, polymerase, template complexity), this calculator also provides a small suggested gradient window around the recommended value.
Interpreting the results
The results table shows each primer’s length, GC percentage, and estimated Tm. The pair Ta column repeats the recommended annealing temperature derived from both primers. Rows are highlighted when basic checks suggest the primer may be outside typical ranges.
Practical tips
- Use a gradient PCR if you see nonspecific bands or low yield - try several temperatures around the recommended value.
- Keep primer pairs balanced - a large Tm gap can reduce performance and may require redesign.
- Watch GC extremes - very high GC can increase secondary structures and reduce specificity.
Limitations
This is an estimation tool. For high-precision design, more advanced thermodynamic models and exact buffer conditions are often required. Still, the Wallace rule plus a small gradient range is a practical starting point for many standard PCR setups.