Calculating the melting temperature of PCR primers

Calculating an accurate melting temperature of your oligos, your template and of the predicted product is important to set the cycling parameters of your PCR machine.

To test a pair of primers, use Primer Design (Primer3).

  • Open your template sequence
  • Run ANALYZE > PRIMER DESIGN/TEST (Pairs)
  • Click on the Use This Primer checkbox for both the left and the right primer
  • Add your primers
  • To test a single primer use Quicktest Primer

  • Open your template sequence
  • Run ANALYZE > QUICKTEST PRIMER (Individual)
  • Paste in your primer sequence
  • QuickTest Primer shows Santa Lucia, Breslauer and Baldino estimates of the Tm.

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    We always strive to use the most up to date algorithms to calculate Tm:

    MacVector uses the combined thermodynamic parameters of Santa Lucia (PNAS 95:1460-1465, 1998) which have become widely accepted as the most accurate values.
    MacVector makes adjustments for Mg++ and dNTP concentrations in the reaction mixture as described by (Ahsen et al, Clinical Chemistry 47:1956-1961, 2001).

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