DNA Melting Temperature Calculator

Formula Used

1) Wallace rule for short oligos:
Tm = 2 × (A + T) + 4 × (G + C)

2) GC long-oligo estimate:
Tm = 64.9 + 41 × (GC count − 16.4) / length

3) Salt-adjusted estimate:
Tm = 81.5 + 16.6 × log10[Na⁺] + 0.41 × GC% − 500 / length

4) DMSO correction:
Corrected Tm = Recommended Tm − (0.6 × DMSO%)

5) Suggested annealing temperature:
Annealing = Corrected Recommended Tm − user-defined offset

The calculator recommends the Wallace rule for very short oligos and the salt-adjusted formula for standard primer lengths. These equations are useful screening estimates, not a substitute for full nearest-neighbor thermodynamic modeling.

How to Use This Calculator

Enter a label for the sequence if you want named exports.
Paste a DNA sequence using only A, T, G, and C.
Enter sodium concentration in mM for ionic correction.
Enter DMSO percentage if your reaction includes DMSO.
Choose an annealing offset, commonly around 3 to 5 °C.
Press the calculate button to view results above the form.
Review the recommended Tm, annealing estimate, notes, and graph.
Use the CSV or PDF buttons to export the result.

Sequence	Length	GC%	Wallace Tm (°C)	GC Long Tm (°C)	Salt-Adjusted Tm (°C)
ATGCATGCATGC	12	50.00	36.00	29.37	38.73
GCGCGCGCTAAT	12	66.67	40.00	36.20	45.57
ATATATATATAT	12	0.00	24.00	8.87	18.23

FAQs

1) What is DNA melting temperature?

DNA melting temperature is the point where half of a DNA duplex becomes single stranded. It estimates duplex stability under defined solution conditions and helps guide primer design, probe selection, and annealing choices.

2) Why does GC content increase Tm?

GC pairs form three hydrogen bonds, while AT pairs form two. Higher GC content usually strengthens duplex stability, which raises the temperature needed to separate the two strands.

3) Why does salt concentration matter?

Positive ions shield negative phosphate charges on DNA backbones. Better shielding reduces strand repulsion, improves duplex stability, and generally increases melting temperature.

4) When should I trust the Wallace rule?

The Wallace rule is most useful for short oligos and quick estimates. It is simple and fast, but longer primers and complex reaction conditions usually need more detailed formulas.

5) How does DMSO affect melting temperature?

DMSO often lowers melting temperature because it weakens base pairing and reduces duplex stability. That is why the calculator subtracts a correction from the recommended Tm when DMSO is entered.

6) Is annealing temperature the same as Tm?

No. Annealing temperature is typically set a few degrees below the primer melting temperature. The exact offset depends on primer design, polymerase system, and experimental optimization.

7) Do mismatches change the result?

Yes. Base mismatches can significantly reduce duplex stability and lower actual melting temperature. Simple formulas do not fully capture that effect, so experimental verification is still important.

8) Is this calculator enough for final assay design?

It is excellent for screening and planning, but final assay design should also consider hairpins, dimers, mismatches, Mg2+, buffer composition, and laboratory validation.