How to Use the DNA Melting Temperature Calculator
Step 1: Enter your DNA sequence in the input box. Works best for primer-length sequences of 10 to 60 bases.
Step 2: Set your reaction conditions — Na⁺ concentration (default 50mM) and primer concentration (default 250nM).
Step 3: Click Calculate Tm. Two values are shown — Wallace rule for quick estimation and nearest neighbour for more accurate results.
Step 4: Use the recommended annealing temperature (Tm − 5°C) as your starting PCR annealing temperature and optimize from there.
Understanding Melting Temperature
The melting temperature (Tm) is the temperature at which 50% of DNA molecules are in double-stranded form and 50% are in single-stranded form. It is one of the most important parameters for PCR primer design.
Tm = 2°C × (A + T) + 4°C × (G + C)
// Basic formula (for sequences 14-50 bp):
Tm = 81.5 + 16.6 × log[Na⁺] + 0.41 × GC%
− 675/length
// Nearest Neighbour method:
More accurate — considers base stacking interactions
Tm = ΔH / (ΔS + R × ln(CT/4)) − 273.15
Corrected for salt concentration
Which Method to Use?
For sequences shorter than 14 bases use the Wallace rule. For primers of 15 to 60 bases the nearest neighbour method is more accurate. For very long sequences neither method is ideal — they are designed for primer-length oligos.
Optimal Primer Tm Range
The ideal primer Tm for standard PCR is between 50°C and 65°C. Primers with Tm below 45°C may not bind efficiently. Primers above 70°C may cause non-specific binding. For best results keep both forward and reverse primer Tm values within 5°C of each other.