Online DNA GC Content & Tm Calculator — GC%, Base Composition, Molecular Weight
GC content determines duplex stability: G-C pairs form three hydrogen bonds while A-T pairs form only two, so higher GC content means a higher melting temperature. Primer design typically targets a GC% of 40–60%.
Tm (melting temperature) — two empirical formulas are provided here, each suited to a different length range; do not mix them up:
- Wallace rule, for short oligonucleotides ≤14 nt:
Tm = 2×(A+T) + 4×(G+C) - GC empirical formula, for approximately 14–50 nt:
Tm = 64.9 + 41×(G + C − 16.4) / N
An important caveat: both are empirical estimates that do not account for salt concentration, primer concentration, or nearest-neighbor thermodynamic parameters. For setting a PCR annealing temperature, use the nearest-neighbor algorithm provided by your primer supplier or run a gradient PCR. This tool is intended for rapid elimination of obviously unsuitable candidate sequences.
Molecular weight is calculated for single-stranded DNA (including the 5′ phosphate) as the sum of individual nucleotide molecular weights minus (n−1) water molecules.
FAQ
The two Tm values differ — which one should I use?
It depends on sequence length. Use the Wallace rule for sequences ≤14 nt and the GC empirical formula for 14–50 nt. Both are estimates; for setting a PCR annealing temperature, rely on a nearest-neighbor algorithm or gradient PCR results.
Why does my primer pass the GC% check but still fail to amplify?
GC% is only the coarsest filter. You should also check whether the 3′ end forms a hairpin, whether primer dimers are likely, and whether the primer maps uniquely to the template. This tool does not perform those checks.
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