Primer Designer

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

References for PCR primer design methods: nearest-neighbor primer Tm calculations, salt and magnesium correction, Primer3-style primer screening, restriction overhang sequence handling, and IUPAC degenerate DNA codes. Mol Biology Tools implementation code is original.

  1. Primer Tm calculations: SantaLucia J. (1998), unified nearest-neighbor DNA thermodynamics.
  2. Salt and magnesium correction for DNA duplex Tm: Owczarzy et al. (2008), DNA duplex stability in magnesium and monovalent cation solutions.
  3. Primer-design screening heuristics: Untergasser et al. (2012), Primer3 design constraints and primer-quality checks.
  4. Degenerate DNA and reverse-complement nomenclature: NC-IUB/IUPAC nucleotide ambiguity recommendations.