DNA/RNA Melting Temperature (Tm) Calculator
Determine the melting temperature (Tm) of oligonucleotide primers. Input a sequence to analyze composition, GC content, molecular weight, and salt-adjusted Tm.
Sequence Analysis
Enter primer sequence and adjust reaction conditions.
Melting Temperature (Tm)
* Salt-adjusted Tm: 48.2 °C.
Expert Tip
For optimal PCR results, choose primers between 18-30 bp in length with a GC content of 40-60%. Make sure the Tm of the forward and reverse primers differ by less than 5°C.
Methodology & Equations
Wallace Rule
Suitable for short primers ($< 14$ bases). Estimates Tm based on hydrogen bond count:
Basic Salt-Adjusted
Preferred for medium-length oligonucleotides ($\ge 14$ bp) under standard laboratory salt conditions:
Advanced Salt Correction
Corrects for sodium and magnesium ion concentration equivalents, affecting double-stranded primer stability:
How to Calculate DNA/RNA Melting Temperature (Tm)
The melting temperature (Tm) is the temperature at which 50% of the oligonucleotide duplex dissociates:
Analyze Nucleotide Composition
Count A, T, G, C bases in the sequence.
Choose Wallace Method (< 14 bp)
Use the basic equation: Tm = 2 × (A+T) + 4 × (G+C).
Choose Salt-Adjusted Method (≥ 14 bp)
Use: Tm = 81.5 + 16.6 × log10([Na+]) + 41 × (G+C)/Length - 500/Length, adjusting for salt concentration.
Related Calculators
View All Biology Tools →Annealing Temperature
Compute the optimal annealing temperature for primers using basic or salt-adjusted Tm.
PCR Master Mix
Calculate reactant volumes for multi-well PCR reactions.
DNA Concentration
Compute DNA/RNA sample concentration from A260.