PCR Primer Calculator (Tm & GC%)

What this calculator reports

• Tm (Nearest-Neighbor): SantaLucia 1998 with Ceff and optional 16.6·log₁₀[Na⁺]; adjusted for DMSO/formamide.
• Tm (Wallace) and Tm (GC% model): quick estimates for sanity checks.
• GC content, length, and GC clamp quality near the 3′ end.
• Quick self-structure heuristics: longest 3′ self-complement and internal complement (hairpin stem proxy).

For advanced Mg²⁺ corrections (Owczarzy) or RNA NN parameters, consider a future pro mode. This page aims to be fast and friendly for routine PCR primer checks.

PCR Primer Basics: Tm, GC% and Annealing Tips

This PCR Primer Calculator estimates melting temperature (Tm) and GC content so you can quickly sanity-check candidate primers before synthesis. Tm represents the temperature at which half of the primer–template duplex is denatured. A higher Tm generally indicates a more stable duplex. GC% is the fraction of bases that are G or C; because G≡C pairs form three hydrogen bonds and stack strongly, GC-rich primers typically have higher Tm than AT-rich primers of the same length.

Which Tm should I trust?

The calculator shows three values: Wallace (very quick, short oligos), a GC% empirical estimate, and an nearest-neighbor (NN) Tm using SantaLucia-style thermodynamics. For actual PCR setup, the nearest-neighbor Tm with your real salt concentration and primer concentration is the best guide, because it sums per-dimer enthalpy/entropy and applies a salt correction. Remember that solvents such as DMSO and formamide lower Tm; the calculator applies practical offsets so you can plan annealing temperature accordingly.

Design guidelines that save time

• Length: 18–24 nt is a common starting range for PCR. Very short primers can bind nonspecifically; very long primers can form secondary structures.
• GC%: Aim for roughly 40–60%. Extreme GC or AT content often yields poor amplification or off-target binding.
• 3′ GC clamp: Having 1–3 G/C within the last five 3′ bases helps stable extension without over-stabilizing mis-matches.
• Runs & repeats: Avoid homopolymer runs ≥5 (e.g., AAAAA) and simple repeats that can slippage-prime.
• Self-structure: Minimize 3′ self-complementarity (self-dimer) and internal complementarity (hairpins). The quick heuristics here flag long complementary stretches.
• Forward/Reverse balance: Keep primer Tm values within ~2–3 °C of each other so a single annealing step works for both strands.
• Amplicon size: For standard PCR, 100–1000 bp is typical. For qPCR, 70–200 bp is common to improve efficiency and sensitivity.

Choosing an annealing temperature (Ta)

A practical starting point is Ta ≈ Tm(NN) − 3–5 °C. If you observe non-specific bands, try a gradient PCR to refine Ta. Increasing monovalent salt (Na⁺/K⁺) or magnesium (Mg²⁺) stabilizes duplexes and raises effective Tm, while DMSO/formamide lower it. This page applies a simple monovalent salt term; for Mg²⁺-heavy conditions, more advanced corrections (e.g., Owczarzy) are recommended.

Good hygiene before ordering

• Clean sequences: Restrict to A/C/G/T; remove spaces and numbers. Degenerate bases should be used deliberately (they broaden specificity and may reduce effective concentration).
• Specificity checks: After you like the Tm/GC% profile, validate predicted off-targets with genome-aware search tools for your organism.
• Report & share: Use the copy button to capture Tm (NN), GC%, length, clamp quality, and structure flags for your lab notes.

Keywords: PCR primer calculator, primer Tm, GC content, nearest-neighbor DNA Tm, Wallace rule, annealing temperature, salt concentration, DMSO, formamide, self-dimer, hairpin, GC clamp, primer design.