PCR Primer Tm Calculator Formula
Understand the math behind the pcr primer tm calculator. Each variable explained with a worked example.
Formulas Used
Tm (Basic Formula)
tm_basic = 64.9 + 41 * (gc_count - 16.4) / lengthTm (Salt-Corrected)
tm_salt = 81.5 + 16.6 * log10(na_conc / 1000) + 41 * gc_frac - 675 / lengthSuggested Annealing Temp (Tm-5)
annealing = 81.5 + 16.6 * log10(na_conc / 1000) + 41 * gc_frac - 675 / length - 5Variables
| Variable | Description | Default |
|---|---|---|
length | Primer Length(bp) | 22 |
gc_count | Number of G+C Bases | 12 |
na_conc | Na+ Concentration(mM) | 50 |
gc_frac | Derived value= gc_count / length | calculated |
at_count | Derived value= length - gc_count | calculated |
How It Works
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.
Worked Example
A 22-mer primer with 12 GC bases at 50 mM Na+.
length = 22gc_count = 12na_conc = 50
- 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
Ready to run the numbers?
Open PCR Primer Tm Calculator