Laboratory Mechanism of Genotyping Fruit Flies (Genetics) Part 1

 Module I: Isolation of Drosophila DNA

In this portion of the experiment, the DNA of the common fruit fly (Drosophila melanogaster) is isolated for later use. Initially, two 0.5 mL screw-cap tubes were labeled with the designation of “wild” and “white” separately. Once the tubes were labeled, wild type and white eyed fruit flies were transferred via vials and anesthetized in the freezer for 3 minutes. Additionally, four anesthetized wild type flies were transferred into the tube labeled “wild”, where the flies were mashed for approximately 10 seconds. Once mashed, 100 microliters of prepared Lysis buffer is added to the tube, via a micropipette with an unused tip. Moreover, the sample was mixed by pipetting up and down, once mixed the sample tube was placed into an ice chest. The process done for the wild type sample, was repeated for the white eyed fruit flies. Thus, when both tubes were mashed and iced, they were placed into a water bath of 70o C for 15 minutes of incubation. After the process of incubation, both samples received 14 microliters of potassium acetate and were mixed for approximately 5 seconds. Finally, the samples were placed back into the ice chest for approximately 5 - 10 minutes, then for a duration of 5 minutes the samples were centrifuged at maximum speed. As a result, the supernatant of each sample was removed and placed into two unused 0.5 mL microcentrifuge tubes. It is important to note, that in removing the supernatant, the pellet within the tubes is not disturbed. Additionally, 45 microliters of room temperature isopropanol was incorporated into each of the samples. As a result, the DNA in the samples is allowed to precipitate. Regardless, the samples were required to centrifuge at maximum speed for 5 minutes, after the addition of the isopropanol. Consequently, due to the centrifugation, a small DNA pellet was formed at the bottom of the tubes: a marker was utilized to indicate the location of the pellets. Therefore, the removal of the supernatant was needed. However, this time after removing the supernatant, 20 microliters of 70% ETOH was utilized to wash the pellet of each sample. Again, the samples were requiring another centrifugation for 4 minutes at the maximum speed. Alongside, another process of removing and discarding the supernatant. Once accomplished, the pellet was left to dry for approximately 5-10 minutes. The addition of 25 microliters of Universal DNA Buffer was utilized to resuspend the DNA pellet of each sample. Lastly, the samples were placed back into the ice chest, to complete the last step of Module I of the laboratory process.

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