Annealing Temperature Calculator
Estimate a PCR primer’s melting temperature and a suggested annealing temperature from its length and GC content.
What This Calculator Does
This tool estimates the melting temperature (Tm) of a PCR primer and suggests a starting annealing temperature for your PCR program, based on primer length and GC content.
The Method
Short primers (under 14 bases) use the classic Wallace rule: Tm = 2×(A+T) + 4×(G+C). Longer primers use the more accurate GC-content formula: Tm = 64.9 + 41×((GC count − 16.4) / length). The suggested annealing temperature is set 5°C below the calculated Tm, a common starting point for optimization.
- Primer length should be the total number of bases in your primer.
- GC content is the percentage of G and C bases in the sequence.
These formulas give a useful starting estimate for general lab planning and educational purposes. Actual optimal annealing temperature can vary with salt concentration, primer secondary structure, and polymerase used, so gradient PCR is recommended to fine-tune real reactions.