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Tools

Sequence tools

Paste a DNA, RNA or protein sequence: reverse complement, six-frame translation and open reading frames, GC content, restriction sites, protein mass and extinction coefficient, and primer melting temperatures.

Your sequence

Primer melting temperature

Reaction conditions

SantaLucia (1998) nearest-neighbour parameters with the salt correction used by Primer3 (Mg²⁺ converted to a sodium equivalent as in von Ahsen et al. 2001). Values from other calculators differ by a few degrees when they use other parameters or concentrations.

Notes on the methods

  • Translation uses the standard genetic code (NCBI table 1). Mitochondrial and some microbial genomes use other codes.
  • Open reading frames run from ATG to the first stop codon. Real genes may start at other codons, contain introns, or be shorter than the longest ORF.
  • Primer Tm uses SantaLucia’s 1998 nearest-neighbour values with the salt correction of Primer3, so it is close to Primer3 and Primer-BLAST with the same concentrations. Polymerase suppliers’ calculators often use other values; follow your polymerase’s guide for the annealing temperature.
  • Protein parameters follow ExPASy ProtParam: average masses and Pace’s extinction coefficients. Measured ε can differ by a few per cent.

References

  1. SantaLucia J Jr. 1998. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. PNAS 95:1460–1465. doi:10.1073/pnas.95.4.1460
  2. von Ahsen N, Wittwer CT, Schütz E. 2001. Oligonucleotide melting temperatures under PCR conditions: nearest-neighbor corrections for Mg²⁺, deoxynucleotide triphosphate, and dimethyl sulfoxide concentrations. Clinical Chemistry 47:1956–1961. doi:10.1093/clinchem/47.11.1956
  3. Untergasser A, et al. 2012. Primer3—new capabilities and interfaces. Nucleic Acids Research 40:e115. doi:10.1093/nar/gks596
  4. Pace CN, Vajdos F, Fee L, Grimsley G, Gray T. 1995. How to measure and predict the molar absorption coefficient of a protein. Protein Science 4:2411–2423. doi:10.1002/pro.5560041120
  5. Gasteiger E, et al. 2005. Protein identification and analysis tools on the ExPASy server. In: The Proteomics Protocols Handbook, 571–607. Humana Press. doi:10.1385/1-59259-890-0:571
  6. Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, Cormier MJ. 1992. Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111:229–233. (The example sequence, ENA/GenBank M62653.1.) doi:10.1016/0378-1119(92)90691-H