Skip to content
ENExplore Calculators

Primer Tm Calculator

Estimate primer melting temperature and recommend an annealing temperature.

About Primer Tm Calculator

Melting temperature rises with GC content because guanine and cytosine form three hydrogen bonds rather than two. Salt concentration stabilises the duplex, which is why the buffer composition shifts Tm measurably. Primer pairs should have Tm values within about two degrees of each other, because a single annealing temperature has to work for both.

Formula

Primer Tm

Tm = 64.9 + 41 × (GC − 16.4) ÷ length, with salt and concentration corrections

Tm
Temperature at which half the duplex has separated
annealing temperature
Usually set about 5 degrees below Tm

Worked example

With the default values below, Primer Tm Calculator returns Melting Temperature of 38.84. Change any input above and every figure here is recalculated in your browser.

Inputs used

Primer lengthaccepts 6–60 bases
20 bases
Number of G and C basesminimum 0 bases
10 bases
Monovalent salt concentrationminimum 1 mM
50 mM
Primer concentrationminimum 1 nM
250 nM

Results

Melting Temperature
38.84 C
GC Content
50.00%
Recommended Annealing Temperature
33.84 C
Basic Formula Melting Temperature
51.78 C
Primer Quality Note
GC content and length are in the usual working range

Step by step

  1. 1Values used
  2. 2Primer Tm
  3. 3Melting Temperature= 38.84 C
  4. 4GC Content= 50.00
  5. 5Recommended Annealing Temperature= 33.84 C
  6. 6Basic Formula Melting Temperature= 51.78 C
  7. 7Primer Quality Note= GC content and length are in the usual working range

Assumptions and limits

  • The result is derived only from the 4 inputs listed above. Anything not among them is not part of the calculation.
  • In particular, this calculator does not take inflation, tax, fees or charges or compounding frequency into account.
  • Figures are rounded for display to 2 decimal places, so totals built from several rounded parts can differ slightly from the unrounded arithmetic.

How these calculators are built and tested is described on the Methodology page. If a figure looks wrong, please report it.

Frequently Asked Questions

How is Primer Tm calculated?

Tm = 64.9 + 41 × (GC − 16.4) ÷ length, with salt and concentration corrections Melting temperature rises with GC content because guanine and cytosine form three hydrogen bonds rather than two. Salt concentration stabilises the duplex, which is why the buffer composition shifts Tm measurably.

Why does Primer Tm matter?

Primer pairs should have Tm values within about two degrees of each other, because a single annealing temperature has to work for both.

What values do I need to enter?

This calculator takes 4 inputs: Primer length, Number of G and C bases, Monovalent salt concentration, Primer concentration. The pre-filled defaults are a realistic starting point — replace them with figures from your own environment for a result you can act on.

Related Calculators

Primer Tm Calculator | Calcrivo