Primer Designer

Loading tool…

Design forward and reverse PCR primers from a DNA template with this free online PCR primer design tool, using target melting temperature or primer length, optional restriction enzyme overhangs, and downstream binding-site and amplicon checks.

Primer design inputs
No sequence loaded.

Accepted file types: .txt, .fa, .fasta, .fna, .ffn, .seq, .gb, .gbk, .genbank. SnapGene .dna files are not yet supported; export as FASTA or GenBank.

Use this sequence in another tool

Transfer the current validated sequence to another Mol Biology Tools page in a new tab.

Amplicon and primer settings

Leave amplicon region blank to use the full sequence. Circular templates can use ranges that cross base 1, such as 4800-250. Enter either primer length or target Tm.

Optional cloning additions

Each primer accepts one restriction site only. Search by enzyme name or recognition sequence, or type one exact DNA/IUPAC motif. Auto flanking uses built-in enzyme-specific rules with at least 3 bases; if no rule is available, it falls back to GCGC.

Advanced thermodynamic settings

References

Sources for primer Tm, DNA sequence formats, and restriction-enzyme data. Primer selection and structure screening use original Mol Biology Tools code, not the Primer3 engine.

  1. Perfect-match binding-region Tm: SantaLucia (1998), nearest-neighbor DNA thermodynamics. Includes terminal initiation and self-complementarity corrections.
  2. Salt and magnesium correction: Owczarzy et al. (2008), mixed-salt duplex stability. Free Mg2+ is approximated as total magnesium minus total dNTP, with a minimum of zero.
  3. Primer-design background: Untergasser et al. (2012), Primer3. Background reading, not the implementation used here; hairpin and dimer scores are screening estimates.
  4. Recognition sites and cleavage offsets: NEB recognition-sequence chart. The shared enzyme snapshot, verified 14 September 2026, retains individual source links.
  5. Compatible restriction enzyme ends: New England Biolabs compatible cohesive ends chart.
  6. IUPAC notation for restriction motifs: NC-IUB/IUPAC nucleotide ambiguity recommendations (1985). Ambiguity codes apply to custom enzyme motifs; the input template must contain A, C, G and T.
  7. Sequence-file formats: NCBI FASTA documentation and GenBank sample record.