Primer Tm Calculator

About Primer Tm Calculator

This tool estimates the melting temperature of a DNA primer three ways: nearest-neighbor thermodynamics, an empirical GC-percentage formula and the Wallace rule. You set primer and sodium concentration, and it reports each estimate side by side so you can see how far the quick formulas drift from the thermodynamic model.

How to use it

1. Paste the primer sequence into the Primer sequence box. Length and GC percentage appear as badges below it.
2. Enter Primer conc. in micromolar. The default is 0.25.
3. Enter [Na+] in millimolar. The default is 50.
4. Compare the three tiles: Nearest-neighbor, GC% method and Wallace rule.

A 17-nt sequencing primer

GTAAAACGACGGCCAGT (0.25 uM primer, 50 mM Na+)

Nearest-neighbor 51.6 C, GC% method 47.1 C, Wallace rule 52 C, 53% GC

When to use it

• Matching the melting temperatures of a forward and reverse primer pair before ordering.
• Picking a starting annealing temperature, commonly a few degrees below the lower primer Tm.
• Seeing how much the estimate moves when salt or primer concentration changes.
• Teaching why length and GC content drive duplex stability.

Questions about Primer Tm Calculator

Which nearest-neighbor model is used?

The unified 1998 SantaLucia parameter set. It sums enthalpy and entropy over each adjacent base pair, adds terminal initiation terms, applies a monovalent salt correction to entropy, and assumes a non-self-complementary primer in excess.

Does it account for magnesium or dNTPs?

No. Only sodium and primer concentration are included. Real PCR buffers contain Mg2+, which raises the effective Tm, so treat the figure as a comparison between primers rather than the exact annealing temperature.

Why does the nearest-neighbor tile show a dash?

It needs at least two bases, no ambiguity codes, and positive primer and salt concentrations. A degenerate base or a zero value leaves the tile empty, though the other methods still calculate.

Which method should I trust?

Use nearest-neighbor for ordinary primers. The GC formula is meant for longer oligos, and Wallace only for very short ones, so both can disagree with it noticeably.

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