Free PCR Primer Tm Calculator
Calculate PCR primer melting temperature with salt concentration correction for annealing temperature selection.
bp
mM
Tm (Basic Formula)
56.7 °C
Tm (Salt-Corrected)51.6 °C
Suggested Annealing Temp (Tm-5)46.6 °C
Tm (Basic Formula) vs Primer Length
PCR Primer Melting Temperature
Accurate Tm prediction is essential for PCR primer design. The salt-corrected formula accounts for ionic strength effects on DNA stability.
Salt-Corrected Formula
Tm = 81.5 + 16.6 × log10([Na+]) + 41 × (GC fraction) - 675/N
where [Na+] is in moles/L and N is the primer length. The annealing temperature for PCR is typically set 3-5°C below the primer Tm.
Example Calculation
A 22-mer primer with 12 GC bases at 50 mM Na+.
- 01GC fraction = 12/22 = 0.5455
- 02Tm = 81.5 + 16.6 × log10(0.050) + 41 × 0.5455 - 675/22
- 03Tm = 81.5 + 16.6 × (-1.301) + 22.36 - 30.68
- 04Tm = 81.5 - 21.60 + 22.36 - 30.68 = 51.58°C
- 05Annealing temp ≈ 51.58 - 5 = 46.58°C
Frequently Asked Questions
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