This annealing temperature calculator estimates the temperature to run the annealing step of a PCR from a single primer sequence, along with the primer's melting temperature and GC content. Getting the annealing temperature right is one of the most important parts of designing a reliable PCR: set it too high and the primer will not bind, so you get no product, while setting it too low lets the primer stick in the wrong places, giving faint or spurious bands. The calculator works from the base composition of the sequence you paste in. It counts the A, T, G and C bases, then estimates the melting temperature (Tm), the point at which half of the primer-template duplex has come apart. For short primers up to about 13 bases it uses the Wallace rule, which adds 2 degrees for every A or T and 4 degrees for every G or C. For longer primers, which covers most that you design, it switches to the GC-content formula that scales with the proportion of strong G and C pairs. It then applies the widely used rule of thumb that the annealing temperature sits about 5 degrees below the Tm. Enter your primer, read off the suggested annealing temperature, and treat it as a starting point: when a primer pair has mismatched melting temperatures, work from the lower one, and run a gradient PCR to fine tune the best setting for your template and polymerase.
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46.8°C
suggested annealing temperature (Ta)
Melting temperature (Tm)51.8°C
GC content50.0%
Primer length20 bp
Ta is estimated as Tm minus 5 degrees. Tm uses the Wallace rule for primers of 13 bases or fewer and a GC-content formula for longer primers. Estimate only: optimise with a gradient PCR.
How it works
The calculator counts the four bases in your primer. For a primer of 13 bases or fewer it uses the Wallace rule, Tm = 2 x (A + T) + 4 x (G + C). For longer primers it uses the GC-content formula, Tm = 64.9 + 41 x (G + C - 16.4) / length, where length is the total number of bases. The annealing temperature is then Tm - 5. GC content is the share of G and C bases, which raises the melting temperature because those pairs are held together by three hydrogen bonds rather than two.
Worked example
Take the 20-base primer AGCTTGGCATCGATCCTAGA. It contains 5 A, 5 T, 5 G and 5 C, so there are 10 G or C bases and the GC content is 50.0%. As it is longer than 13 bases, the Tm is 64.9 + 41 x (10 - 16.4) / 20, which is about 51.8 degrees Celsius. The suggested annealing temperature is 51.8 - 5, which rounds to 46.8 degrees. That is the temperature to start with for the annealing step, adjusting up if you see non-specific products.
