While these rules of thumb offer a general approximation of Tm, there are better algorithms available that are free and easy to use, and that will provide an extremely accurate Tm estimation. We offer one such algorithm for free use in our SciTools® OligoAnalyzer®
application (see http://www.idtdna.com
). This detailed equation takes into account the effects of sodium and magnesium on Tm calculations. These cations have a significant impact on the functional Tm of an oligonucleotide in solution due to their charge. For the most accurate Tm estimation we recommend entering your sequence and adjusting the ionic concentrations in the OligoAnalyzer tool so that they match your reaction conditions. You can view the exact algorithm used for our Tm calculations by clicking on the Definitions link in the OligoAnalyzer program.