The preferences used to calculate the results and their importance are described below:
The value of nucleic acid concentration (C) is the concentration of the target sequence. Since it keeps on changing empiricaly with the advent of reaction, its value is set virtually based on the experimental data4 . It is therefore advised not to change this value unless experimental conditions are very uncommon. This value is used for calculating melting temperature of primer.
This preference should be set to the sum of the concentrations of all the monovalent ions present in the reaction mixture.
This preference should be set to the concentration of Mg2+ ions, used as binders, in a reaction mixture.
This value is calculated using the value of the Monovalent ion concentration and Free [Mg2+] ion concentration. The formula for calculating Total [Na+] equivalent is as follows:
Total [Na+] equivalent = [Monovalent ion concentration] + 4 X sqrt (Free [Mg2+] ion concentration X 1000)
This value is used as salt concentration for calculating the melting temperature of the primer.
This preference should be set to the ambient room temperature. It is used to calculate various DG values as given in theories and formulas. DG is calculated using the formula DG = DH - T * DS.