Annealing temperature calculator.
Find the annealing temperature for a PCR primer. Paste the primer sequence and the calculator works out its melting temperature, GC content, and a suggested annealing temperature.
Primer sequence
LiveTm uses the GC formula, 64.9 + 41 x (GC – 16.4) / length, for primers of about 14 bases or more. Annealing temperature is set 5 C below Tm.
Annealing temperature
55.0 C
suggested, 5 C below the Tm
An estimate; optimal annealing temperature also depends on salt, primer concentration and the polymerase. Calculations run in your browser; nothing is stored.
How it works
From sequence to temperature
A primer’s melting temperature, Tm, is the temperature at which half of it is bound to its template. It rises with length and with GC content, since G-C pairs have three hydrogen bonds to A-T’s two. The calculator counts the bases in your sequence and applies the GC formula.
The annealing temperature for PCR is then set a few degrees below the Tm, commonly about 5 C below the lower of the two primers, so the primers bind specifically without falling off.
Reference
Celsius to annealing.
As a guide, the annealing temperature is set about 5 C below a primer pair’s lower melting temperature.
| Primer Tm | Annealing (Tm – 5) |
|---|---|
| 55 C | 50 C |
| 58 C | 53 C |
| 60 C | 55 C |
| 62 C | 57 C |
| 65 C | 60 C |
The full guide
The complete guide to annealing temperature.
What Tm and annealing temperature are, how they are estimated, and how to use them in PCR.
Tm and annealing temperature
The melting temperature, Tm, is where half of a primer is bound to its target. The annealing temperature is the temperature you actually run in the PCR annealing step, set a little below the Tm so the primer binds tightly and specifically.
Too low an annealing temperature lets primers bind in the wrong places, giving non-specific products; too high and they do not bind at all, so nothing amplifies.
How Tm is estimated
For short oligos the Wallace rule, 2 times A plus T plus 4 times G plus C, gives a quick estimate. For typical primers of 14 bases or more, the GC formula, 64.9 plus 41 times (GC minus 16.4) divided by length, is more reliable because it accounts for length.
Both ignore salt and primer concentration, so they are estimates; nearest-neighbour models are the most accurate but need software.
Setting the annealing temperature
A common starting point is 5 C below the lower primer Tm. If your primers have different Tms, use the lower one so both bind. From there, a temperature gradient across a few degrees finds the cleanest result.
Design primer pairs with similar Tms, ideally within a couple of degrees, so a single annealing temperature suits both.
Designing good primers
Aim for 18 to 25 bases, 40 to 60% GC, and a Tm around 55 to 65 C. Avoid long runs of one base, strong self-complementarity and a 3-prime end that is too GC-rich. Keep the pair’s Tms close.
Use this calculator on each primer, then set the annealing temperature from the lower Tm and optimise with a gradient if needed.
The formula
Bind, then
extend.
Tm rises with length and GC. The GC formula estimates it, and annealing is set about 5 C below.
More lab tools ›# Melting temperature (GC formula)
Tm = 64.9 + 41 × (GC − 16.4) / length
# Annealing temperature
Ta = Tm − 5Questions
Annealing questions.
How do I calculate annealing temperature?
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Find the primer’s melting temperature, then subtract about 5 C. The calculator estimates Tm from the sequence with the GC formula and suggests an annealing temperature 5 C below it.
How is primer Tm calculated?
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For primers of about 14 bases or more, the GC formula is 64.9 plus 41 times (GC count minus 16.4) divided by length. Shorter oligos use the Wallace rule, 2(A+T) + 4(G+C).
What is a good annealing temperature?
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Usually 5 C below the lower primer Tm, often landing around 50 to 60 C. Run a temperature gradient to find the cleanest, most specific product.
Why does GC content matter?
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G-C base pairs have three hydrogen bonds versus two for A-T, so higher GC content raises the melting temperature and makes the primer bind more tightly.
Is this annealing temperature calculator free?
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Yes. It is completely free with no sign-up, and every calculation runs locally in your browser, so nothing you enter is stored or sent anywhere.
About the developer
Jean Borg
Jean builds and maintains every calculator on freecalculators.pro from Malta, with a focus on tools that are fast, free and show their working. The annealing temperature calculator uses the GC melting-temperature formula and is for lab planning and education.