# PCR Primer Tm Calculator (Wallace / Nearest-Neighbor)

Primer melting temperature by Wallace's 2-4 rule and the SantaLucia 1998 unified nearest-neighbor model (16 stacking parameters, terminal initiation, salt correction at your primer/[Na+] concentrations), plus GC content and suggested Ta = Tm − 5°C. Derived from Wallace 1979, SantaLucia 1998, Primer3 2012. Primer-screening use only.

> Canonical page: https://elysiatools.com/en/tools/pcr-primer-tm

- **Category:** Education

- **Keywords:** primer tm calculator, melting temperature, wallace rule, nearest neighbor tm, santalucia thermodynamics, primer design, gc content, annealing temperature, pcr primer, oligonucleotide tm, primer3, pcr

## Overview

Calculate a PCR primer’s melting temperature (Tm) from a 5–60 nucleotide DNA sequence using the Wallace rule or the SantaLucia 1998 nearest-neighbor model. The tool also reports GC content and a suggested annealing-temperature starting point of Tm − 5°C.

## Inputs

- **Primer Sequence (5'→3')** (text): DNA primer sequence, A/C/G/T only (case-insensitive); 5–60 nt.
- **Tm Method** (select): Wallace for a quick short-oligo estimate; nearest-neighbor is the standard for primer design.
- **Primer Concentration (nM)** (number): Primer strand concentration (nearest-neighbor method only).
- **Sodium Concentration (mM)** (number): \[Na+\] of the reaction buffer (nearest-neighbor method only).
- **Decimal Places** (number)

## When to use

- Estimate Tm during initial PCR primer screening.
- Compare the quick Wallace estimate with the nearest-neighbor result.
- Check GC content and account for primer and sodium concentrations with the nearest-neighbor method.

## How it works

- Enter a DNA primer sequence in the 5′→3′ direction using only A, C, G, and T, with 5–60 nucleotides.
- Choose Wallace rule for a quick estimate or SantaLucia nearest-neighbor for a thermodynamic calculation.
- For nearest-neighbor calculations, enter the primer concentration and sodium concentration of the reaction buffer.
- Review the selected Tm, the other reported Tm value, GC content, sequence length, and suggested Ta start.

## Use cases

- Screening candidate forward and reverse PCR primers before laboratory testing.
- Comparing a simple Wallace estimate with a concentration- and salt-adjusted nearest-neighbor Tm.
- Reviewing primer GC content and selecting an initial annealing-temperature range.

## Frequently asked questions

### What sequence format does the calculator accept?

Enter a 5–60 nt DNA sequence in the 5′→3′ direction using only A, C, G, and T. Input is case-insensitive.

### Which Tm methods are available?

The calculator supports Wallace’s 2°C·AT + 4°C·GC rule and the SantaLucia 1998 unified nearest-neighbor model.

### Which method is better for primer design?

Nearest-neighbor is the standard choice for primer design because it considers sequence stacking, terminal initiation, concentration, and salt.

### Do primer and sodium concentrations affect both methods?

No. These concentrations are used by the nearest-neighbor method only. Wallace uses base counts without concentration or salt correction.

### What does the suggested Ta value mean?

It is a suggested annealing-temperature starting point calculated as the selected Tm minus 5°C. Use it for initial screening, not as a final PCR setting.

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