Enter your DNA Sequence (5' to 3'), Primer Concentration, Salt Concentration, Mg²⁺ Concentration, and preferred Calculation Method to find your Melting Temperature (Tm), along with your sequence's GC Content, Sequence Length, Molecular Weight, and a Suggested Annealing Temperature to help you nail your PCR conditions.
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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Suggested Annealing Temperature
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Tm is the temperature at which 50% of DNA strands dissociate from their complementary strands. It's crucial for designing PCR primers and optimizing reaction conditions.
Nearest Neighbor is more accurate as it considers base pair interactions and thermodynamic parameters. Basic method uses simple rules and is less precise but faster for rough estimates.
Higher salt concentrations increase Tm by stabilizing DNA duplexes. Typical PCR reactions use 50-100 mM monovalent salt concentration for optimal primer annealing.
Generally, use 3-5°C below the calculated Tm. Our calculator suggests an annealing temperature that works well for most PCR applications.
Primer concentration affects the equilibrium between bound and unbound DNA strands. Higher concentrations increase effective Tm, impacting PCR efficiency and specificity.
Higher GC content increases Tm because G-C base pairs form three hydrogen bonds compared to two for A-T pairs, making them more thermally stable.
This calculator is optimized for DNA sequences. RNA has different thermodynamic properties and would require specific RNA Tm calculation parameters.
This calculator assumes perfect complementarity. For primers with mismatches or chemical modifications, consider only the perfectly matched portion for accurate Tm calculation.