Gradient Polymerase Chain Reaction to Determine the Optimum Annealing Temperature

To perform a gradient PCR, start by preparing a master mix. Then, remove 19 microliters of the master mix for the non-template control, or NTC, in a 96-well plate. Then, add the template to the remaining master mix. Aliquot 20 microliters of the sample mix into the 96-well plate. Perform PCR following the cycling parameters, and then generate the thermal melt profiles, using the parameters mentioned in the manuscript.