Primer Melting Temperature (Tm) Calculator
Enter one or two PCR primers to get their melting temperature by three methods, GC content, length, molecular weight and quick design checks, plus a suggested annealing temperature for the pair.
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Optional: PCR product (for the Rychlik optimal annealing temperature)
How to use the Primer Melting Temperature (Tm) Calculator
- Paste your forward primer 5'→3'. Spaces, numbers and 5'/3' labels are ignored, and U is read as T.
- Add the reverse primer (optional) to compare the pair and check for primer-dimers.
- Set the reaction conditions, or pick a preset. Use your polymerase buffer's K⁺/Na⁺ and Mg²⁺ values and the final primer concentration.
- Read the results: the nearest-neighbor Tm is the most accurate; the salt-adjusted and basic values are given for comparison with older protocols.
- Start PCR at the suggested Ta and fine-tune with a gradient. Copy the results for your lab notebook.
The three Tm formulas
| Method | Formula | Best for |
|---|---|---|
| Wallace rule | Tm = 2 °C × (A + T) + 4 °C × (G + C) | Quick estimate for short oligos (under 14 nt) |
| Marmur–Doty (basic) | Tm = 64.9 + 41 × (G + C − 16.4) / N | Rough estimate for 14+ nt at about 50 mM Na⁺ |
| Salt-adjusted | Tm = 81.5 + 16.6 × log₁₀[Na⁺] + 0.41 × %GC − 600 / N | Comparing primers across different monovalent salt levels |
| Nearest-neighbor | Tm = ΔH° / (ΔS° + R × ln(CT/4)) − 273.15 | Most accurate; the recommended value |
N is the primer length, [Na⁺] is in mol/L and CT is the primer concentration. In the nearest-neighbor method, ΔH° and ΔS° are the sums of the SantaLucia (1998) unified parameters for each dinucleotide step plus terminal initiation terms. The entropy is salt-corrected with ΔS° + 0.368 × (N − 1) × ln[Na⁺eq], where Mg²⁺ is converted to a sodium equivalent of 120 × √([Mg²⁺] − [dNTP]) mM. For self-complementary primers CT is used instead of CT/4. Molecular weight uses the anhydrous formula (A × 313.21 + T × 304.2 + C × 289.18 + G × 329.21 − 61.96) for an unmodified oligo with a 5' hydroxyl.
Primer design checklist
| Property | Typical target | Why it matters |
|---|---|---|
| Length | 18–25 nt | Long enough to be specific, short enough to anneal efficiently |
| GC content | 40–60% | Balanced stability; very GC-rich primers bind non-specifically |
| Tm | 55–65 °C | Lets you use a stringent annealing temperature |
| Tm difference in a pair | ≤ 2–3 °C | Both primers anneal well at the same temperature |
| 3' GC clamp | 1–2 G/C in last 5 bases, ideally ending in G or C | Stabilizes the 3' end where the polymerase starts |
| Runs and repeats | No runs of 4+ identical bases or 4+ dinucleotide repeats | Reduces slipping and mispriming |
| Self- and cross-complementarity | No 3'-end complementary stretch of 4+ bases | Prevents hairpins and primer-dimers |
The hairpin and dimer checks here look for contiguous complementary stretches only. They are a quick screen, not a full secondary-structure prediction. To design a reverse primer from the bottom strand of your target, use the DNA Reverse Complement Tool.
Frequently asked questions
What is primer melting temperature (Tm)?
Tm is the temperature at which half of the primer molecules are bound to their complementary DNA and half are free. It depends on the sequence, length, salt concentration and primer concentration. Annealing temperatures in PCR are set relative to Tm.
Which Tm method should I use?
Use the nearest-neighbor value. It adds up the measured stability of each pair of neighboring bases (SantaLucia 1998) and corrects for salt and primer concentration, so it typically lands within a few degrees of measured values. The basic formulas only count G and C bases and can be off by 5–10 °C.
How do I choose the annealing temperature?
A common starting point is about 5 °C below the lower Tm of the two primers, then refine with a temperature gradient. Some high-fidelity polymerases recommend annealing at or above Tm instead, so always check your enzyme's instructions.
Why does my supplier show a different Tm?
Calculators use different parameter sets, salt corrections and default concentrations. A Tm calculated at 50 mM Na⁺ and 50 nM oligo will be several degrees lower than one at 1 M Na⁺ or with Mg²⁺. Enter the same conditions to compare like with like.
How does Mg²⁺ affect Tm?
Divalent magnesium stabilizes DNA duplexes much more than sodium, so a typical 1.5–2 mM Mg²⁺ raises Tm noticeably. dNTPs bind Mg²⁺, so the free amount is roughly Mg²⁺ minus dNTPs. The tool converts free Mg²⁺ to a sodium equivalent (120 × √[Mg²⁺ − dNTP], von Ahsen et al. 2001).
What makes a good PCR primer?
Typical guidelines are 18–25 bases, 40–60% GC, a Tm of about 55–65 °C with both primers within 2–3 °C of each other, one or two G or C bases at the 3' end, and no long runs, repeats or self-complementary stretches.
Can I use degenerate bases like N or R?
Not directly. Tm depends on the actual base, so replace each degenerate position with a specific base. Calculating with the GC-richest and AT-richest choices gives you the range of possible Tm values.
Are all the tools on FreeToolCabin tested for quality and accuracy?
Yes! Every tool on our site undergoes automated Q/A testing in real browser environments to ensure high precision, correct output, and zero errors. If you happen to spot a bug or have a suggestion, please contact us at [email protected] and our team will review it right away.