The melting temperature of an oligonucleotide is calculated with three methods:1)Thermodynamic Tm:The Tm is calculated using the nearest-neighbor thermodynamic values method (Breslauer et al). This method is less accurate for longer oligos. The formula for the Tm isTm=dH/(dS-Rln(Cd))-273.15+16.6*(log10(Cs))Where dH is the enthalpy, dS is the entropy, R is 1.987 cal K-1 mol-1, Cd is the DNA concentration, and Cs is the salt concentration.2)Hybridisation Tm:This method is generally used for DNA or RNA hybridization, especially in presence of high salt and formamide. It is more accurate with longer oligos. The Tm is calculated using the following formula:for DNA:DNA hybridization:Tm=81.5+16.6*(log10[Na+])+0.41*[%(G+C)]-0.63*(%Formamide)-500/L-1.5(%Mismatch)for DNA:RNA hybridization:Tm=79.8+18.5*(log10[Na+])+0.58*[%(G+C)]+11.8*[%(G+C)]2-0.5*(%Formamide)-820/L-1.5(%Mismatch);for RNA:RNA hybridization:Tm=79.8+18.5*(log10[Na+])+0.58*[%(G+C)]+11.8*[%(G+C)]2-0.35*(%Formamide)-820/L-1.5(%Mismatch);* where L is the length (bases) of the oligonucleotide.3)GC+AT Tm: the estimated Tm is the sum of the contribution of each base: 2°C for A and T and 4°C for G and C.There are several parameters involved in Tm calculation.DNA concentration is used only in thermodynamic Tm. You may increase oligo DNA concentration in order to increase Tm.Salt concentration affects thermodynamic Tm and hybridization Tm. In the [Na+][mM] box, you may type a suitable salt concentration of your experiments. The value must be an integer greater than 0.Hybridization type is required for Hybridization Tm. In the DNA/RNA box, enter the hybridization type: