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Dna Sequencing To See Phylogenetic Relationships Between Extant Species

1232 words - 5 pages

The hypotheses tested was questioning evolutionary phylogenetic relationship of extant species today. In this experiment, our studies questioned evolutionary phylogenetic relationships of species among us today. The hypotheses tested was to test where a given animal species is placed on the tree of life, but my personal hypotheses was questioning where pig is placed on the tree of life and if my results would compare to it’s placement on the tree of life known today.
In part one of the experiment, we isolated animal DNA, and ran PCR to amplify the molecular characters among animals that bare key traits, which represent key states, in animal evolution. To do so, we had to obtain a sample of ...view middle of the document...

Then, in the new tube, we added 3 µL of silica resin, and then mixed by pipetting up and down. After mixing we incubated the tube for 5 minutes in a water bath then heat block at 57°C. Silica resin is a white, DNA binding matrix that, when in the presence of lysis solution, binds easily to nucleic acids. After this incubation, we placed our tubes in a balanced micro centrifuge, centrifuged for 30 seconds at maximum speed to pellet the resin. The centrifugation pellets the silica resin, now bound to nucleic acid, and it appears as a tiny tear-shaped particle on the bottom of the tube. Then, we used a new-tipped micropipette to remove supernatant without disrupting the white silica resin pellet at the bottom of the tube. Next, we added 500 µL of ice-cold wash buffer to the pellet in the tube, closed the tube, and mixed the tube to re-suspend the silica resin, then placed our tubes in a balanced microfuge for 30 seconds at maximum speed to pellet the resin. After pelleting, we removed the supernatant with a fresh-tipped micropipette without disrupting the pellet at the bottom of our tubes. After centrifuging, we repeated the above steps once more. We used wash buffer, containing ethanol, to remove contaminants from the sample, while leaving nucleic acids bounded to the resin, as silica resin not soluble in the wash buffer. Silica resin may stay as a pellet or it may break up while washing. Washing two times is more effective than one wash with twice the volume of wash, so we performed two washes. After the two washes, we added 100 µL of distilled water to the silica resin and mixed by inverting the tube, because nucleic acids are removed from the silica resin in the presence of water after wash buffer has been removed, and incubate the mixture at 57°C for 5 minutes. After incubation, we centrifuged the tube for 30 seconds at maximum speed to form a pellet of resin. Then, we took a new 1.5 mL tube with our ID, and transferred 90 µL of the supernatant to the tube without disturbing the pellet. For PCR, we took 2 µL of DNA from our supernatant, 10 µL of reaction mix (Gotaq polymerase, buffer), 8 µL COI primers (corresponding to animal choses - my primer was one of the ones for all vertebrates) and put these all in the tube with freshly-tipped micropipettes each time, brought the tubes to PCR machine and began PCR reaction. The PCR conditions included 1 cycle of 94 °C for 1 minute, 35...

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