How do we estimate the Tm of our oligonucleotides?
The melting temperature (Tm value) of an oligonucleotide is dependent upon the length of the sequence, the G+C content of the sequence, and the type and concentrations of cation present, particularly sodium ion, Na+. A variety of formulas have been used for predicting Tm values. The formula that we used for the Tm is the following: Tm = 81.5C + 16.6C x (log10[Na+] + [K+]) + 0.41C x (%GC) 675/N, where N is the length of the oligo. The one that we used takes into account the salt concentration of the reaction, as PCR is typically performed in the presence of ~50mM monovalent cations.
