The high base solution will rupture the cell membrane and create pores in the membrane. Then 100µl of 0.1M HCl and 100µl of 1M Tris – HCl was added at a pH of 7. The cell organelles are present in the solution and they are separated from the solution by centrifugation. The crude cell lysate will contain the DNA. From the DNA, the required gene can be isolated using the PCR analysis.The forward and reverse primers were chosen for PER3 gene and they were added to the PCR tubes along with crude DNA, polymerase buffer, Taq polymerase, and dNTPs. Forward and reverse primers are chosen based on the sequence of the PER3 gene. The primers must have the unique sequence region of each gene that is not present in any other gene. The chance for primer-dimer formation is high because of many reasons. The annealing temperature may not be correct or the template may not be proper or the DNA may have contaminants. To prevent the primer – dimer formation, the above points must be taken care off. DNA polymerase buffer helps to increase the dNTPs binding with the DNA template. Taq polymerase enzyme polymerizes double-stranded DNA formation. It acts as the catalyst for the amplification reaction.The master mix was prepared according to the instructions. The tubes were then placed in the PCR and the amplification was performed. To check the success of PCR amplification, the amplified PCR product was run on the agarose gel electrophoresis.2% the agarose gel was prepared and cast in the casting tray. The electrophoresis box was filled with the electrophoresis running buffer and the DNA products were loaded in the gel with the loading dye. DNA ladder was loaded in the first lane and the samples in the other lanes. The positive control was a readily available one and the negative control was distilled water. The positive control enables us to make sure that the separation of the DNA bands is according to the PER 3 gene amplification or not. DNA ladder is added to the first lane because; they show the bands with the fixed molecular weight.
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