NEB Tm Calculator

Estimate the melting temperature (T_m) of your PCR primers and oligonucleotides instantly. Paste one or two sequences below and this calculator returns the T_m using the nearest-neighbor thermodynamic model (SantaLucia, 1998), with salt and oligo-concentration corrections, plus two classic estimates for comparison.

How to use this T_m calculator

1. Paste your primer (5’→3′) into the first box. Add a second primer to compare a forward/reverse pair side by side.
2. Set the conditions. Default values are 500 nM oligo and 50 mM monovalent salt [Na⁺], which are typical for standard PCR. Adjust them to match your reaction.
3. Read the result. The large number is the nearest-neighbor T_m — the most accurate estimate. Length and GC% are shown alongside, and the table lists the alternative methods.

Only A, C, G and T are read; spaces, numbers and line breaks are ignored, so you can paste sequences straight from a chromatogram or order sheet.

What is the melting temperature (T_m)?

The melting temperature is the temperature at which half of the oligonucleotide duplexes are dissociated into single strands. It is the single most useful number when designing PCR, because the annealing temperature of a reaction is chosen relative to the primer T_m. As a rule of thumb, a starting annealing temperature is set roughly 3–5 °C below the lower of the two primer T_m values, although the exact recommendation depends on the polymerase and buffer you use (see the note on NEB polymerases below).

T_m depends on more than just the sequence. It rises with higher salt concentration (cations stabilise the negatively charged backbone) and with higher oligo concentration, and it falls as the duplex gets shorter or more AT-rich. That is why a reliable estimate has to take the reaction conditions into account, not just count bases.

Calculation methods

Nearest-neighbor (SantaLucia, 1998) — recommended

The nearest-neighbor (NN) model is the modern standard used by tools such as Primer3 and IDT OligoAnalyzer. Instead of treating bases independently, it sums the enthalpy (ΔH) and entropy (ΔS) contributions of each adjacent base pair, adds initiation terms, and applies a salt correction:

Tm = (1000 · ΔH) / (ΔS + R · ln(CT/x)) − 273.15

where R is the gas constant (1.987 cal·mol⁻¹·K⁻¹), CT is the total strand concentration, and x is 4 for non-self-complementary oligos. A monovalent-salt term, 0.368 · (N−1) · ln[Na+], corrects the entropy for the buffer. This is the value shown as the main result.

Salt-adjusted GC% method

For longer templates, a fast approximation based on GC content and salt is:

Tm = 81.5 + 16.6 · log10[Na+] + 0.41 · (%GC) − 600/length

Basic (Wallace) rule

For short oligos (roughly ≤14 nt) the classic Wallace rule gives a quick estimate: Tm = 2·(A+T) + 4·(G+C). It ignores salt and concentration, so use it only as a sanity check.

How this relates to the NEB T_m Calculator

New England Biolabs provides a widely used T_m Calculator that recommends an annealing temperature for a specific NEB polymerase or master mix (Q5, Phusion, OneTaq, Taq, LongAmp, and others). Those recommendations build on the same nearest-neighbor thermodynamics used here, but each enzyme has its own buffer chemistry and its own annealing-temperature guidance.

Use the calculator on this page for an instant, general-purpose T_m while you design or troubleshoot primers. When you are ready to run a reaction with a particular NEB enzyme, confirm the recommended annealing temperature with the official NEB T_m Calculator, since it applies enzyme-specific corrections.

Worked example

Take the primer GTAAAACGACGGCCAGT (17 nt, 53% GC). At 500 nM oligo and 50 mM Na⁺, the nearest-neighbor model returns a T_m of about 53 °C. A reasonable starting annealing temperature for a standard reaction would therefore be around 48–50 °C, to be refined with a temperature gradient if you see weak or non-specific amplification.

Tips for a reliable T_m

• Match the conditions to your reaction. Entering the actual salt and primer concentration matters far more than the choice of formula.
• Design pairs with similar T_m. Keep the two primers within ~5 °C of each other so a single annealing temperature works for both.
• Aim for 40–60% GC and a length of 18–24 nt for most applications.
• Mind the polymerase. High-fidelity enzymes such as Q5 and Phusion often use higher annealing temperatures than Taq for the same primers.

Annealing temperature calculator

The annealing temperature (Ta) of a PCR is derived from the primer T_m. A common rule of thumb is Ta = (the lower primer T_m) − 3 to 5 °C. Use the calculator above to get the T_m of both primers, take the lower value, and subtract a few degrees as a starting point — then fine-tune with a temperature gradient if you see weak or non-specific product. The optimal Ta also depends on which polymerase you use (see below).

Q5, Phusion and Taq: polymerase-specific T_m

High-fidelity polymerases use different buffers and annealing guidance than standard Taq, so the recommended annealing temperature for the same primers varies by enzyme:

• Q5 / Q5 Hot Start: NEB’s Q5 model often recommends a higher annealing temperature than Taq.
• Phusion: high-fidelity guidance; the annealing temperature is enzyme-specific.
• Taq / OneTaq: standard guidance, roughly 3–5 °C below the lower primer T_m.

The calculator here gives a general nearest-neighbor T_m for designing and comparing primers. For the exact, enzyme-specific annealing temperature for Q5, Phusion, OneTaq, Taq and other NEB enzymes, use the official NEB T_m Calculator, which applies the polymerase-specific corrections.

Frequently asked questions

What annealing temperature should I use?

A common starting point is 3–5 °C below the lower primer T_m. For NEB Q5 and Phusion, follow the enzyme-specific value from the official NEB T_m Calculator, which can differ from this rule of thumb.

Why does my T_m differ from another tool?

Different tools use different thermodynamic tables, salt corrections and default concentrations. Make sure the oligo and salt concentrations match before comparing. This calculator uses the unified SantaLucia (1998) parameters.

Which method is most accurate?

The nearest-neighbor model is the most accurate for typical primers and is shown as the main result. The GC% and Wallace estimates are provided only for quick comparison.

Does the calculator account for salt and primer concentration?

Yes. The nearest-neighbor result applies a monovalent-salt correction and uses your oligo concentration, both of which shift the T_m.

Can I calculate the T_m of two primers at once?

Yes. Enter a second sequence to see both T_m values side by side, which is the quickest way to check that a forward/reverse pair is balanced.

What is the annealing temperature for Q5 or Phusion?

It is enzyme-specific and usually higher than for Taq. Get the primer T_m here, then confirm the exact Q5 or Phusion annealing temperature with the official NEB T_m Calculator.

Related guide: How to design PCR primers →