DNA Melting Temperature (Tm) Calculator

Calculate DNA duplex melting temperature from sequence GC content and length.

Number of G+C bases (bases)

Number of A+T bases (bases)

Primer/oligo length (bp) (bp)

For short oligos, use Wallace rule

Method

Please check your inputs and try again.

Worked Examples

Example 1: 20bp primer: 12 G/C, 8 A/T

Wallace: 4×12+2×8

Result: 64°C

Typical PCR primer Tm

Example 2: High-GC primer 25bp: 18GC, 7AT

Basic formula

Result: 66.8°C annealing 61.8°C

GC-rich primer — high Tm

Frequently Asked Questions

What annealing temperature to use in PCR?

Start with Tm − 5°C for initial optimization. For gradient PCR, try Tm − 10°C to Tm + 3°C. Higher annealing temp → more specificity; lower → more product but more non-specific bands.

Why does GC content affect Tm?

G≡C pairs form 3 hydrogen bonds; A=T pairs form only 2. More GC content requires more energy to separate strands, raising Tm. Each % increase in GC raises Tm by ~0.41°C.