A PCR kit (#333) was purchased from Edvotek. The protocol provided with this kit was used with modification from the Edvotek lab manual. DNA samples were obtained from human buccal cells from individuals in a class using two cotton-tipped applicators. The DNA sample on the applicator was placed in a tube with two milliliters of Phosphate Buffer Solution (PBS). DNA was released into the tube by twirling and rubbing against the applicators walls of the tube to release as many cells as possible from the swabs. This step was done for approximately thirty seconds. The DNA sample was centrifuged at 5000-6000g for no longer than one minute. After centrifugation, the supernatant was removed using a pipette and the pellet was combined with the chelating agent. It was vigorously inverted back and forth multiple times and pipetted in and out multiple times with the use of a calibrated transfer pipet. The chelating agent was loosened and one hundred microliters of the liquid was transferred into the tube containing the white pellet for resuspension. The tube was vortexed and the cells were lysed in a boiling water bath for ten minutes followed by cooling. The sample was centrifuged for approximately two minutes resulting in the supernatant containing the DNA. Twenty to fifty microliters of the supernatant was removed and placed into a 0.5 milliliter tube and was set aside on ice to cool while the PCR reaction was prepared.
The PCR reaction pellet was mixed with 20.0 microliters of PV92 primer solution and 5.0 microliters of the supernatant. The sample was centrifuged and dissolved and ready for the PCR cycle.
The control was performed by the instructor using the cheek cell DNA that was provided in