Tm Calculator (Melting Temperature)

The Tm Calculator finds the melting temperature of a DNA or RNA sequence — the temperature at which half the double-stranded nucleic acid separates into single strands, a critical value for designing PCR primers and hybridization experiments. Enter your DNA/RNA sequence, select a calculation method (Wallace Rule or Nearest Neighbor), and provide your primer concentration, Na⁺ salt concentration, and optional Mg²⁺ concentration to get your melting temperature (Tm) in °C. Secondary outputs include GC content, sequence length, and molecular weight.

Enter nucleotide sequence using standard bases: A, T, G, C for DNA or A, U, G, C for RNA

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mM
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Results

Melting Temperature (Tm)

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GC Content

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Sequence Length

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Molecular Weight

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Frequently Asked Questions

What is melting temperature (Tm) and why is it important?

Melting temperature (Tm) is the temperature at which 50% of DNA double strands dissociate into single strands. It's crucial for PCR primer design, hybridization assays, and determining optimal annealing temperatures.

What's the difference between basic and nearest neighbor methods?

The basic method (Wallace rule) uses simple base counting rules, while the nearest neighbor method considers thermodynamic parameters of adjacent base pairs for more accurate predictions, especially for longer sequences.

How does salt concentration affect melting temperature?

Higher salt concentrations stabilize DNA duplexes and increase Tm. Na+ and Mg2+ ions shield negative charges on DNA backbone, making it easier for strands to remain paired at higher temperatures.

What primer concentration should I use for PCR?

Typical PCR primer concentrations range from 0.1 to 1.0 μM, with 0.2-0.5 μM being most common. Higher concentrations can increase Tm but may also promote primer-dimer formation.

How accurate are Tm calculations compared to experimental values?

Nearest neighbor calculations typically predict Tm within 2-5°C of experimental values under standard conditions. Accuracy depends on sequence length, salt conditions, and absence of secondary structures.

Can this calculator handle RNA sequences?

Yes, the calculator can process RNA sequences. Simply use U instead of T in your sequence. The thermodynamic parameters will be adjusted accordingly for RNA-RNA hybridization.

What sequence length works best for Tm calculations?

The calculator works for sequences from 2 to several hundred bases. For PCR primers (15-30 bases), nearest neighbor method gives most accurate results. Very short sequences may have less reliable predictions.

How do I choose the right annealing temperature for PCR?

Generally, use an annealing temperature 2-5°C below the calculated Tm. For primer pairs, use the lower Tm minus 2-5°C. Start with Tm-5°C and optimize if needed.