Mitochondrial DNA Lab Report

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Introduction When observing a human population there are many phenotypical differences such as; behavior, development, and morphology. Not only are there phenotypical differences amongst a human population, but there are also genotypic differences that can be observed using DNA sequencing data. One way to observe genetic variation amongst humans would be to sequence the Mitochondrial DNA(mtDNA). Mitochondrial DNA is the most commonly used DNA to form phylogenetic trees. Human Mitochondrial DNA is about 16,500 bps in size and consists of 13 protein coding genes, 22 tRNAs (transfer RNA), and 2 rRNAs (ribosomal RNA) (Ingman, 2001). Mitochondrial DNA has many beneficial uses for DNA sequencing such as; its ability to be extracted from small …show more content…
First, students swabbed the inside of their cheek for at least 30 seconds with a cotton swab to obtain their sample for DNA extraction. 180 µl of Buffer ATL and 20 µl of proteinase K were added to the swabbed sample using sterile micropipetting techniques. The swabbed samples were incubated at 56 ⁰C in a hot block. Next, students isolated their HV1 control region DNA through a series of washes which contained 200µl of Buffer ATL, 200µl of ethanol, 500 µl of Buffer AW1 and AW2, and 50 µl of Buffer AE (Penn State Biology 220W Lab Manual, pgs. 48-49) After completing a series of washes, students quantified the amount of DNA in their sample using the NanoVue Spectrophotometer. Using their results from the NanoVue, students were able to determine the amount of their sample DNA and PCR master mix they needed to add to PCR tubes. Then, the samples were loaded into the PCR machine to amplify DNA samples. Once the PCR cycle was complete, samples were stored in a freezer at -20⁰C. With the products from PCR, students used Gel Electrophoresis to separate electrically charged molecules. Gel Electrophoresis requires 3 steps; preparing a gel solution, gel electrophoresis, and photographing the gel. To prepare the gel, students mixed .3g of agarose and 30mLs of 1X TAE buffer in a 125 ml flask (Penn State Biology 220W Lab Manual, pgs.55-59). The mixture was microwaved and set to cool for 3 minutes. After the cooling process, 1µl of ethidium bromide dye was added to the agarose. The mixture was then poured into the gel caster of the electrophoresis unit. Once the mixture formed a solid gel, 1X TAE buffer was poured over the electrophoresis unit. Students loaded 6 µl of their samples into the wells and watched as the blue tracking dye migrated down the gel. Once the dye migrated half way down the gel, gels were placed on a

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