How to calculate primer Tm and GC content

The melting temperature is the temperature at which half the DNA strands separate — critical for setting PCR annealing temperatures. For short primers (14 bases or fewer) the simple Wallace rule works well: T_m = 2 × (A + T) + 4 × (G + C), because G-C pairs have three hydrogen bonds and A-T pairs only two. For longer sequences use T_m = 64.9 + 41 × (G + C − 16.4) / N, where N is the length. GC content is just (G + C) / N × 100. The reverse complement is what you get by pairing each base (A↔T, G↔C) and reading the new strand 5′→3′ — the sequence of the opposite strand, and the basis for designing a reverse primer.

Method note: these are the standard teaching formulas (basic and salt-independent). Real primer design tools also account for salt and oligo concentration using nearest-neighbor thermodynamics, which shifts T_m by a few degrees.

Related tools: DNA → protein translation · all biochem tools.