GC Content / Tm Calculator
Calculate GC content and PCR primer melting temperature
Tool Guide
Definition
GC content & primer Tm calculator — Computes GC percentage and melting temperature (Tm) of a DNA sequence. Wallace rule: Tm = 2(A+T) + 4(G+C) (good for 14–20 nt). Nearest-neighbor method: sums per-dinucleotide ΔH/ΔS for higher accuracy (Primer3 standard).
Purpose
(1) PCR primer design — keep Tm difference between Fwd/Rev pair ≤ 5°C (2) Determine annealing temperature — typically Tm − 5°C (3) Check qPCR probe (TaqMan) GC% 40–60% (4) Verify sequencing primer / cloning oligo Tm
How to Use
① Enter primer/oligo sequence (case-insensitive) ② Auto-displays: • Length (nt) • GC content (%) • Tm (Wallace, °C — for short primers) • Tm (nearest-neighbor, °C — most accurate) ③ Shows recommended ranges (Tm 50–65°C, GC 40–60%) Nearest-neighbor uses standard conditions (50 mM Na⁺, 250 nM oligo). Tm may vary ±2°C with different buffers.
Examples
Example 1) Forward primer 5'-ATGGCCAAGGTGGACCTG-3' (18 nt) → GC = 11/18 = 61% → Tm (Wallace) = 2×7 + 4×11 = 58°C → Tm (NN) ≈ 60°C → Recommend annealing 55°C Example 2) Reverse primer 5'-CAGGTCCACCTTGGCCAT-3' (18 nt) → GC = 61%, identical Tm → well-matched pair