DNA Melting Temperature Calculator
Estimate the melting temperature (Tm) of a DNA oligonucleotide from its sequence and salt concentration. Enter one strand (A, C, G, T) and the Na⁺ concentration to get the Tm and GC content.
Estimate the Melting Temperature of a DNA Oligonucleotide
DNA Melting Temperature Formula
Where:
- \(A, T, G, C\) — number of adenine, thymine, guanine and cytosine bases in the sequence
- \(n\) — total number of bases (sequence length)
- \([\mathrm{Na^+}]\) — total monovalent salt concentration in mol/L
The first equation is the Wallace rule, used for short oligonucleotides up to 13 bases; the second is the salt-adjusted formula (Howley et al., 1979), applied automatically from 14 bases onward. The calculator picks the appropriate formula for you.
DNA Melting Temperature – Calculation Example
Example: a 15-base oligonucleotide with the sequence ATGCATGCGATTACA in a buffer containing 50 mM Na⁺. The sequence contains 5 A, 4 T, 3 G and 3 C bases, so \(G + C = 6\), \(A + T = 9\) and \(n = 15\).
\[ [\mathrm{Na^+}] = 50\ \mathrm{mM} = 0.05\ \mathrm{mol/L}, \quad \log_{10}(0.05) = -1.301 \]
\[ T_m = 100.5 + 41 \cdot \frac{6}{15} - \frac{820}{15} + 16.6 \cdot (-1.301) \]
\[ T_m = 100.5 + 16.4 - 54.67 - 21.60 = 40.6\ \mathrm{°C} \]
Result: the melting temperature is \(T_m \approx 40.6\) °C and the GC content is 40.0%. The simple Wallace rule would give 2 × 9 + 4 × 6 = 42 °C, but it is not recommended for sequences of 14 bases and longer.
How to Use the Calculator
- Type or paste your sequence into the DNA Sequence (5′→3′) field. Only the letters A, C, G and T are accepted; case, spaces and line breaks are ignored.
- Enter the total monovalent salt concentration in the Salt (Na⁺) Concentration field, in mM (default 50 mM). This value is used only when the sequence has 14 bases or more.
- Read the Melting Temperature (Tm) in °C and the GC Content in percent in the results panel.
- Check Calculation Details to see the base composition, which formula was applied and the substituted numbers, so you can verify the result by hand.
What Is the Melting Temperature?
The melting temperature, or Tm, is the temperature at which half of the DNA duplexes in a solution are dissociated into single strands and half remain paired. It is a direct measure of duplex stability: the higher the Tm, the more heat is needed to separate the two strands. The Tm matters whenever two complementary DNA strands must find and bind each other reliably — designing PCR primers, choosing hybridisation probe conditions, or planning annealing temperatures for short oligos.
Duplex stability comes from the base pairs themselves. A G–C pair is held by three hydrogen bonds while an A–T pair has only two, so GC-rich sequences melt at higher temperatures. In the Wallace rule this is expressed very directly: each A–T pair adds roughly 2 °C to the Tm and each G–C pair roughly 4 °C. The GC content reported alongside the Tm tells you how the sequence is composed — a quick sanity check, since GC content and Tm always rise and fall together.
Salt also plays an important role. Sodium ions shield the negative charges on the phosphate backbone, so a higher Na⁺ concentration stabilises the duplex and raises the Tm. This is why the salt-adjusted formula includes the term \(16.6\,\log_{10}[\mathrm{Na^+}]\): every tenfold increase in salt adds about 16.6 °C to the predicted Tm. When comparing oligos, always compare them at the same salt concentration, and remember that the salt concentration must be entered in mM — the conversion to mol/L is done automatically.
Accuracy, Assumptions and Practical Tips
- Both formulas assume a perfect duplex: the sequence must hybridise to a fully complementary strand with no mismatches, bulges or gaps. Even a single mismatch can lower the true Tm by several degrees.
- Wallace rule limits: it is a rough rule of thumb intended for roughly 4–13 bases near 50 mM Na⁺. It ignores salt concentration and sequence length effects, so use it only for quick estimates on very short oligos.
- Salt-adjusted formula limits: it accounts for length and monovalent salt but not for oligo concentration, terminal effects, denaturants (formamide, DMSO) or divalent cations such as Mg²⁺, which significantly stabilise duplexes in PCR buffers.
- For exact work, use the nearest-neighbour model: modern primer-design software uses nearest-neighbour thermodynamics, which considers the stacking energy of every dinucleotide step. The two formulas here are quick approximations accurate to a few degrees — ideal for planning and cross-checking, not for final assay design.
- Interpreting the result: as a rule of thumb, hybridisation or annealing works best about 5 °C below the Tm. If the calculated Tm is far from your target, adjust the primer length or GC content rather than the annealing temperature alone.
If you work with peptides or proteins in the same experiments, you may also find our Isoelectric Point Calculator useful for planning electrophoresis conditions, and the Buffer Capacity Calculator helps you check whether your hybridisation buffer holds its pH during heating.
Frequently Asked Questions
Why do different Tm calculators give different values for the same sequence?
Different calculators use different models. Simple rules (Wallace, basic salt-adjusted formulas) ignore sequence order, while the nearest-neighbour model accounts for the stacking energy of each dinucleotide step, so it typically returns values several degrees different. Corrections for Mg²⁺, oligo concentration and DMSO also shift the result. Always compare Tm values produced by the same method — mixing Tm values from different calculators when designing primers is a common mistake.
What is a good GC content for a primer or oligonucleotide?
Aim for 40–60% GC content. Primers in this range bind strongly enough to be specific yet still denature and anneal reliably. GC-rich regions above 65% can create stable secondary structures and need higher annealing temperatures, while AT-rich primers below 40% may bind weakly or non-specifically. Also avoid long runs of a single base (e.g. GGGGG) and a 3′ end with many G or C bases, which promotes non-specific priming.
How do I choose the PCR annealing temperature from the Tm?
A common starting point is to set the annealing temperature about 3–5 °C below the lower Tm of the primer pair. If you get non-specific bands, raise the annealing temperature in 1–2 °C steps; if you get no product, lower it slightly. Modern protocols often use gradient PCR or a two-step touchdown programme to find the optimum empirically, since simple Tm formulas only approximate the true behaviour in a PCR buffer.
Can I use this calculator for RNA sequences or sequences containing U?
No. The calculator only accepts the DNA bases A, C, G and T — RNA sequences with uracil (U) are rejected. RNA/DNA and RNA/RNA hybrids are also more stable than DNA/DNA duplexes, so the Wallace and salt-adjusted formulas would underestimate their Tm even after substituting U with T. For RNA oligos, use a nearest-neighbour calculator with RNA thermodynamic parameters instead.
How do Mg²⁺ and DMSO affect the calculated Tm?
Both formulas here assume only monovalent salt, so they underestimate the Tm in typical PCR buffers. Mg²⁺ stabilises duplexes strongly, often raising the effective Tm by several degrees, while denaturants like DMSO or formamide lower it (roughly 0.6 °C per 1% DMSO). If your buffer contains Mg²⁺, treat the result as a conservative estimate and optimise experimentally, or use a nearest-neighbour tool that accepts divalent cation concentrations.
What happens to the Tm if my oligo has a mismatch with its target?
The calculator assumes a perfectly complementary duplex, so it cannot account for mismatches. Even a single mismatch, especially in the middle of the sequence or near the 3′ end of a primer, can lower the true Tm by 5–15 °C and reduce amplification efficiency. For degenerate primers or probes targeting variable regions, use a nearest-neighbour tool that scores each mismatch type separately, and test conditions experimentally.
Do I enter the salt concentration in mM or mol/L, and how do I convert?
Enter the Na⁺ concentration in mM; the calculator converts it to mol/L internally for the logarithm term \(16.6\,\log_{10}[\mathrm{Na^+}]\). To convert by hand, divide mM by 1000: 50 mM = 0.05 mol/L, 500 mM = 0.5 mol/L. Remember that this is the total concentration of monovalent cations — some protocols quote ionic strength or include Tris and K⁺, which should be added to the Na⁺ value for a realistic estimate.