DNA/RNA Melting Temperature (Tm) Calculator

DNA & RNA Melting Temperature Explained

This calculator estimates the melting temperature (Tm) of DNA or RNA sequences, which is the point where about half of the strands are paired and half are separated. Tm is a practical guide for designing primers, planning PCR or qPCR, and checking hybridization conditions for probes. If the temperature is too low, binding can be weak and nonspecific; if it is too high, strands may not anneal at all. A quick Tm estimate helps you choose an annealing temperature and avoid trial-and-error.

The main factors that influence Tm are sequence length, GC content, salt concentration, and strand concentration. G and C bases pair with three hydrogen bonds, so GC-rich sequences tend to melt at higher temperatures than AT- or AU-rich sequences. Longer sequences generally have higher Tm because more base pairs must be disrupted. Ions in the buffer stabilize the duplex, so higher salt typically raises the melting temperature. These effects are captured by different calculation models, which you can compare here.

To use the calculator, paste your sequence, select DNA or RNA, and optionally enter a complementary strand. If you leave Strand B blank, the tool uses the reverse complement of Strand A. Then set the strand concentration and salt conditions. You can also add common solvent adjustments such as DMSO or formamide. The results show Tm values for the available models so you can pick the one that best fits your use case.

• Step 1: Enter a sequence and choose DNA or RNA.
• Step 2: Provide Strand B or let the tool auto-complement Strand A.
• Step 3: Set concentration and salt conditions, then add solvent adjustments if needed.
• Step 4: Review the melting temperature results and compare models.

Models Included

• Wallace rule: Tm = 2 C per A+T/U and 4 C per G+C. Best for very short oligos (under 14 nt).
• GC% empirical: Tm = 64.9 + 41 · ((#G + #C - 16.4) / N), where N is length.
• Nearest-Neighbor (DNA): Tm(K) = ΔH° / (ΔS° + R · ln(C_eff)), with salt correction and symmetry/initiations, then converted to C and adjusted for solvents.

Typical use cases include primer design for PCR, checking a probe for a melt curve experiment, comparing DNA vs RNA stability, or troubleshooting inconsistent amplification. The calculator is also useful in teaching labs where students need to see how GC content or concentration changes Tm. If you are working with very short oligos, start with Wallace or GC% estimates. For longer sequences or more precise work, the nearest-neighbor model is usually the better choice.

RNA Wallace/GC% are computed; RNA nearest-neighbor can be added (hook present).

Notes & Assumptions

• Input cleaning: whitespace and numbers are ignored; DNA uses A/T/C/G, RNA uses A/U/C/G.
• Auto complement: If Strand B is empty, the reverse complement of A is used.
• Concentration: Enter per-strand concentration. For non-self-complementary duplexes, C_eff=C/4 in the NN equation; for self-complementary, C_eff=C/2.
• Salt: Simple monovalent correction 16.6·log₁₀[Na⁺] (M). For Mg²⁺/advanced corrections, a future update can add Owczarzy parameters.
• Solvents: Linear practical offsets (rule-of-thumb): −0.75 °C per 1% DMSO; −0.6 °C per 1% formamide.