🔥 Annealing Temperature Calculator
Estimate primer melting temperatures (Tm) and suggested annealing temperatures for PCR setup.
How to Use This Calculator
Paste or type your primer sequence
Enter a DNA primer sequence using standard nucleotide characters (A, C, G, T). Spaces and hyphens are ignored.
Run the calculation
Click the calculate button to review GC content, melting temperature estimates, and suggested annealing temperature.
Apply in PCR optimization
Use the suggested annealing temperature as a starting point. Fine-tune empirically based on amplification performance.
Formula
Tm (Wallace) = 2 × (A + T) + 4 × (G + C)
For primers longer than 13 nucleotides, a more refined estimate is given by:
Tm (long) = 64.9 + 41 × ( (G + C − 16.4) ÷ N )
A practical annealing temperature (Ta) is typically 3–5 °C below the long-primer Tm. This calculator suggests Ta = Tm (long) − 5 °C by default.
Full Description
PCR primer annealing temperature strongly influences amplification specificity and yield. Primers with balanced length and GC content produce temperatures that reduce off-target binding. This tool supports quick iteration during primer design by reporting GC composition, multiple Tm approximations, and a suggested annealing temperature.
Remember that additives such as DMSO or high salt, as well as primer–template mismatches, can shift the effective annealing temperature. Always confirm optimal conditions experimentally with a gradient PCR or similar optimization strategy.
Frequently Asked Questions
What sequence characters are allowed?
Use standard nucleotides A, C, G, and T. The calculator ignores whitespace and hyphen characters so you can paste formatted sequences.
How accurate are these melting temperatures?
The Wallace rule is ideal for short primers, while the long primer approximation is better for oligos longer than 13 bases. For precise assays, consider nearest-neighbor thermodynamic models and include salt concentration adjustments.
Why is the annealing temperature lower than Tm?
Annealing temperature is typically set a few degrees below the melting temperature to promote specific binding while keeping the primer–template duplex stable during extension.
Can I adjust for salt or additives?
This quick calculator does not incorporate salt corrections. Apply empirically determined adjustments or use a thermodynamic model if you require high-precision estimates.