Calculate melting temperature using basic, salt-adjusted, and nearest-neighbor thermodynamic methods.
Paste or type your primer sequence (5′ to 3′, DNA bases A/T/G/C only). The calculator provides three Tm estimates: the basic Wallace rule (good for short primers <20 nt), the salt-adjusted method (accounts for ionic strength), and the nearest-neighbor thermodynamic method (most accurate, especially for primers 15–30 nt).
For PCR, design primer pairs with Tm values within 2–3°C of each other, and set your annealing temperature 3–5°C below the lower Tm.
Basic (Wallace rule): Tm = 2(A+T) + 4(G+C). Simple count-based formula, only reliable for primers 14–20 nt with ~50% GC content.
Salt-adjusted: Tm = 100.5 + (41 × GC fraction) − (820/length) + 16.6 × log₁₀([Na⁺]). Accounts for primer length and salt concentration.
Nearest-neighbor (SantaLucia 1998): Uses thermodynamic parameters (ΔH and ΔS) for each pair of adjacent bases. Most accurate for primers 15–30 nt. Formula: Tm = (ΔH / (ΔS + R × ln(Ct/4))) − 273.15 + 16.6 × log₁₀([Na⁺]).
GC content: Ideal primer GC content is 40–60%. Primers outside this range may have poor specificity (too low) or strong secondary structures (too high). The 3′ end should ideally have 1–2 G/C bases (a "GC clamp") for efficient extension.