DNA Melting Temperature Calculator

The Temperature Where a Double Helix Falls Apart

Heat a DNA duplex enough and the hydrogen bonds holding its two strands together give way, splitting it into single strands — a process called melting. The melting temperature (Tm) marks the point where half the DNA in a sample has separated, and it depends almost entirely on one thing: how many G-C base pairs are holding the strand together versus A-T pairs, since G-C pairs use three hydrogen bonds instead of two.

The Formula

Short sequences (< 14 bp) — Wallace Rule:
Tm = 4(G+C) + 2(A+T)

Longer sequences (≥ 14 bp) — GC% formula:
Tm = 64.9 + 41 × (GC% − 16.4) ÷ N

The calculator automatically selects the appropriate formula based on sequence length: the Wallace rule for short oligonucleotides like PCR primers, and the GC-content-based formula for longer sequences where each base contributes less individually.

Where This Calculation Matters

PCR primer design — primers need a Tm in a specific range, and closely matched Tm values between forward and reverse primers are essential for efficient amplification.
Hybridization assay conditions — Southern and Northern blot protocols set wash temperatures relative to the probe's calculated Tm.
Molecular cloning — annealing temperatures for oligonucleotide-based cloning steps are chosen relative to Tm.
qPCR probe design — TaqMan and other fluorescent probes require Tm values several degrees above the primers they're paired with.

Tm by GC Content at 100 Base Pairs

Computed from the GC% formula (Tm = 64.9 + 41(GC% - 16.4)/N) at a fixed length of 100 bp
GC Content	Calculated Tm
20%	66.38 °C
40%	74.58 °C
50%	78.68 °C
60%	82.78 °C
80%	90.98 °C

At a fixed length, every 10 percentage points of GC content raises Tm by roughly 4 °C — a useful rule of thumb when scanning primer candidates.

How to Use This Calculator

Enter the DNA Sequence using only the letters A, C, G, and T.
Select Calculate — the calculator automatically applies the Wallace rule for sequences under 14 bp or the GC% formula for longer ones.
Review the returned melting temperature and the formula breakdown used to compute it.

Related Calculations

Studying the enzyme that unwinds and replicates this DNA next? See the Enzyme Rate Calculator. For sizing the resulting protein product, use the Protein Molecular Weight Calculator.