Using the DNA extracted, the MC1R gene was amplified using a Polymerase Chain Reaction. The procedure for performing this section was from “Polymerase Chain Reaction.” The master mix for the PCR was created by adding water, PCR buffer 10x, dNTP mix, forward primer, reverse primer, and Taq polymerase; enough master mix was made for five samples (one extra sample). Two identical PCR samples were made for each group. Each PCR sample consisted of 10 microliters of master mix, 3.38 microliters of 100 ng DNA, and 6.62 microliters of water which were added to a PCR tube. The PCR strip was placed in the thermocycler for amplification.
The next step was to take the MC1R PCR products and perform a gel electrophoresis. The procedure was followed by using “Sample Preparation for Sequencing” The two identical samples were combined, and 12 microliters was added to a sheet of parafilm in addition to 2 microliters of loading dye. To make a point of reference, 7 microliters of DNA ladder was added to one of the columns. The DNA mixture was added to the second column and the gel was run after all the other samples were loaded. The remaining PCR product was then purified from any residual primers using ExoSAP-IT. Three separate tubes were used; 7 microliters of PCR product and 3 microliters of ExoSAP-IT were added to each tube and the tubes were incubated. After incubation, the samples were sent for DNA sequencing.