Primer Tm Calculator and Checker

Work out a primer's melting temperature by the nearest-neighbour method, corrected for salt and magnesium, and check its GC content, 3′ end, runs, hairpins and dimers. Paste the template to see where a pair binds and the size of the product.

Sequence tool

Check a primer

Forward primer, 5′ → 3′

Reverse primer, 5′ → 3′Optional

Primer

Na⁺ (and K⁺)

Mg²⁺

dNTPs, all four

Template (optional)

Bare letters, a FASTA record or a GenBank record. Numbers and spaces from a listing are ignored.

Type a primer, 5′ to 3′, to see its melting temperature and checks. Add the reverse primer and the template for the pair and the product.

The formula

Nearest neighbour

Tm = ΔH ÷ (ΔS + R ln C) − 273.15

Wallace rule, 14 bases or fewer

Tm = 2(A + T) + 4(G + C)

%GC formula

Tm = 81.5 + 0.41 × %GC − 600 ÷ N + 16.6 log₁₀[Na⁺]

ΔH, ΔS: the enthalpy (kcal/mol) and entropy (cal/(mol·K)) of forming the duplex, summed from SantaLucia's (1998) values for each neighbouring pair of bases
R: the gas constant, 1.987 cal/(mol·K)
C: the primer's concentration in mol/L, taken to be far above the template's
[Na⁺], [Mg²⁺]: the salts, which raise the Tm; applied as Owczarzy's (2004, 2008) corrections, with the dNTPs' share of the magnesium taken off
N: the primer's length in bases

Good to know

Compare Tm values only when they were calculated the same way. The nearest-neighbour figure here, at 250 nM primer in 50 mM Na⁺, matches IDT's OligoAnalyzer; Primer3 uses 50 nM and an older salt correction and gives a lower figure.
Add the Mg²⁺ and dNTPs of your reaction to see the Tm in the tube: 1.5 to 2 mM Mg²⁺ raises it by about 6 °C. Set the annealing temperature a few degrees below the lower primer's Tm, and keep the two primers within 5 °C of each other.
The 3′ end matters most. One or two G or C in the last five bases (a GC clamp) hold it down, but more than three make it bind where it shouldn't, and a dimer or hairpin that holds the 3′ end gets extended by the polymerase.
The dimer and hairpin checks count paired bases, not free energy, so they catch the clear cases. For a borderline primer, run it through a free-energy check as well.

In the dictionary: polymerase chain reaction, quantitative PCR and DNA polymerase.

Worked example

The M13 forward (−20) primer, GTAAAACGACGGCCAGT

0117 bases, 9 of them G or C: 52.9% GC
02ΔH = −133.6 kcal/mol, ΔS = −360.7 cal/(mol·K): Tm = 68.6 °C at 1 M Na⁺
03corrected to 250 nM primer in 50 mM Na⁺: 53.6 °C (Wallace rule: 52 °C)

Tm 53.6 °C. On pUC19 it binds at 379 and, with the M13 reverse (−27) primer on the other strand at 465–481, amplifies a 103-bp product across the polylinker.

How it works

The reasoning behind it

A primer melts, or anneals, at the temperature where half of it is paired with its template. The nearest-neighbour method adds up the enthalpy and entropy of each neighbouring pair of bases, because how firmly two bases hold depends on their neighbours as much as on themselves, and turns the sums into a temperature with the primer's concentration in the equation.

Those values are measured in 1 M salt. Real reactions have far less sodium and some magnesium, which screens the charge of the backbone and lets the strands come closer, so the Tm is corrected for both, using Owczarzy's equations, which fit measured melting curves. The dNTPs bind magnesium, so their concentration is taken off first.

The checks look for what makes a primer fail: a 3′ end that is too weak or too strong, runs of one base that let it slip, and stretches that pair with themselves or with the partner primer. Pasting the template shows where each primer sits and how long the product will be.

Questions

Frequently asked

Which Tm should I trust?

The nearest-neighbour value. The Wallace rule is a rough guide for probes of 14 bases or fewer, and the %GC formula ignores the order of the bases. All three are shown so you can recognise the figure a protocol or a supplier quotes.

What annealing temperature should I use?

Start about 3 to 5 °C below the lower of the two primers' Tm values, calculated with the Mg²⁺ of your reaction, and run a gradient if the product is weak or there are extra bands. Some polymerases' suppliers have their own calculators tuned to their buffers; use those for their enzymes.

What is a GC clamp?

A G or C at or near the 3′ end, which pairs more firmly than A or T and helps the polymerase start. One or two in the last five bases is good; more than three makes the end bind stably to the wrong places.

What is a primer dimer?

Two primers paired with each other, or one with itself, by a few complementary bases. If the pairing holds a 3′ end, the polymerase extends it, and the reaction fills with a short product instead of yours. Avoid 3′ ends that pair with each other over more than three or four bases.

Why do IDT, Primer3 and NEB's calculators give different Tm values?

They start from different primer and salt concentrations and use different salt corrections, and NEB's Tm calculator is tuned to its own buffers. Differences of several degrees are normal. This page uses IDT's defaults, so it agrees with OligoAnalyzer under those conditions.

Research

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Each one shows its formula and a worked example, so you can check the answer and learn the method.

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