Denaturation Profile

The nearest-neighbor thermodynamic parameters for DNA pairs in 1 M NaCl were obtained from the SantaLucia and Hicks paper. The molecules of the top and bottom oligos are the same concentration in the hybridization reaction. The model assumes a two-state model, where the single-stranded oligos are in a random coil state, and once annealed, they become double-stranded DNA. The denaturation profiles in 1 M NaCl are plotted over a temperature range from 0 to 100 °C. Enter your sequence in all capital letters and input the total oligos in [nM] for both the top and bottom strands, ensuring equal molarity of complementary oligos (e.g., 250 nM for the top strand and 250 nM for the bottom strand, which gives a total of 500 nM; you are required to input 500 nM in the text field). It will calculate the differences in Gibbs free energy, enthalpy, and entropy between the two states. Hover over the curve to check the melting temperature. Please be advised that this Tm is not directly applicable to your PCR reactions, as it is simulated in 1 M NaCl phosphate buffer. However, BioCal provides the salt-corrected melting temperature (Tm), as recommended by Owczarzy et al., based on the cations present in your preferred PCR buffer.