Enter your DNA or RNA sequence along with your primer concentration, Na+ salt concentration, and Mg2+ concentration, then choose a calculation method to find your melting temperature (Tm) — plus a breakdown of GC content, sequence length, and molecular weight.
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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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.
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.
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.
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.
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.
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.
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.
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.