Rlp Analysis Experiment

Abstract: Given the exuberant number of rape and sexual assault cases and the increased media coverage, it’s important to be able to distinguish between suspects. Can RFLP analysis differentiate between people, specifically in cases of sexual assault and rape on college campuses? To determine this, RFLP analysis performed using students in a BIOL 3600 laboratory. Buccal cells were extracted from the BIOL 3600 students, later purified, and then cut with two restriction endonucleases, MseI and RsaI. The now cut DNA was mixed with a blue loading dye and run on a 1.0% agarose gel (with added TAE buffer and Sybr Safe) at 110 volts. The finished product had bands of diverse base pair lengths, differing from student to student. During the RFLP analysis
The singular deviation during this experiment was that we did not centrifuge the tube (for 5-10 seconds) after incubation. The mitochondrial DNA was amplified in sterile 0.5 ml PCR tubes. The reaction in tube one contained, 2 µl of our genomic DNA, 1 µl JOSH01 (a 10 µM forward primer) and 1 µl JOSH02 (a 10 µm reverse primer, 10 µl 5x Taq and 4 µl MgCl2. 1 µl of Taq polymerase and dNTP as well as 30 µl deionized water were added. For the second tube, 2 µl of JOSH04 (forward primer) and JOSH05 (reverse primer), 4 µl of genomic DNA, 20 µl of 5x Taq reaction buffer, 8 µl MgCl2, 2 µl dNTP mix, 2 µl Taq polymerase enzyme, and 60 µl deionized water were added. Amplification took place at 95⁰C for two minutes for one cycle (initial denaturation) followed by the next three steps happen in thirty cycles, denaturation at a temperature of 95⁰C for one minute, then annealing at 58⁰C for one minute, and extension for two minutes at 72⁰C. At 72⁰C the final extension happens for five minutes at for one cycle. Gel electrophoresis was performed using 1.0% agarose gel electrophoresis, made with TAE and 5 µl of Sybr safe. The PCR products were mixed with loading dye, pipetted at 10 µl and the gel ran for thirty minutes at 110
If it cannot recognize the cut site, then the DNA will not be separated. A singular cut in the DNA fragment will produce two bands, two cuts would create three bands, and so on. In order to properly analyze a gel, the cut sights are used to correctly determine how many restriction enzyme sites were located on each individual DNA strand. Neither Uzma nor Rebekah’s RFLP analysis produced cut sites. However, Sara and Caitlin had two cut sites in both the MseI and RsaI products, producing a total of three bands in each column, meaning that the restriction enzymes each recognized two sites. Sara had banding at 100, 200 and 300 base pairs for her MseI products and banding at 100, 200 and 900 base pairs for her RsaI products. Caitlin however had the same base pair lengths for her MseI and RsaI products, 200, 300 and 1000 base pairs. Sara and Caitlin’s DNA had the same amount of cut sites but at different band lengths, due to the genetic polymorphism within the population of students in the BIOL 3600 lab. This supports the hypothesis that restriction enzymes can recognize different sequences within groups of