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.

The Tm calculations in MacVector were updated in MacVector 12.6 to use a more modern algorithm. MacVector has always used thermodynamic “nearest neighbor” calculations, but there were two changes in MacVector 12.6:

1) MacVector now uses the combined thermodynamic parameters of Santa Lucia (PNAS 95:1460-1465, 1998) which have become widely accepted as the most accurate values.

2) We also now make adjustments for Mg++ and dNTP concentrations in the reaction mixture as described by (Ahsen et al, Clinical Chemistry 47:1956-1961, 2001). These values are now shared by all primer tools in MacVector. However, if you have been using MacVector for many versions it is possible that you will need to “default” the primer settings to clear any older values from the settings.

Additionally the Test PCR Primer Pairs tool was deprecated in MacVector 12.7.5 and it has been removed from MacVector 13. The Design Primers interface in MacVector 13 has new functionality for easily testing a pair of primers. There is nothing that the old tool provides that you cannot do with the MacVector 13.

Quicktest Primer does display the Tm results for both the new (“Santa Lucia”) and older (“Breslauer”) calculations, although to get the same results as MacVector 12.5 you will need to clear the Mg++ and dNTP concentration to 0 (as mentioned above).

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