Laboratory Mechanism of Genotyping Fruit Flies (Genetics) Part 2

 Module II: Amplification of the Extracted DNA

Two PCR tubes were labeled “wild” and “white”, in the beginning of Module II. Additionally, each PCR tube already had a PCR EdvoBead. Regardless, 20 microliters of Primer Mix and 5 microliters of extracted DNA from Module I was added to each tube. Moreover, after the addition of Primer Mix and extracted DNA, the PCR samples were gently mixed. As a result, the samples were observed to have an orange mixture. To collect the samples at the bottom of the tubes, the samples were centrifuged. In addition, to the two samples, a positive control tube was obtained. Lastly, the samples, including the positive control tube were all placed in the ice chest for storage. The experimental process was put on hold for approximately 2 weeks and resumed in

Module III: Separation of PCR products by Electrophoresis

In any case, the protocol is followed thoroughly. However, due to efficiently utilizing time in the lab period, a separate group of students had diluted the concentrated buffer with distilled water. The agarose powder was mixed with the buffer into a 250 mL flask. Boiling the solution, dissolves the agarose powder, which allowed the group to cool the agarose to 600 C. As well as, diluted SYBR Safe stain was incorporated into the agarose. While the agarose was cooling, the ends of the gel-casting tray were secured via tape, and the comb was placed in its appropriate notch. Furthermore, once the agarose solution was cooled to the appropriate amount, the agarose solution was poured into the prepared gel-casting tray. After a few minutes, approximately 20 minutes later, the agarose gel solidified within the gel-casting tray. Lastly, the comb and the tape utilized was removed in prep for electrophoresis. For instance, the gel was placed into the electrophoresis chamber, and the addition of the electrophoresis buffer covered and submerged the gel. On the negative electrode side of the electrophoresis chamber, 25 microliters of the samples were loaded into the wells. Once the samples, including the positive control tube, were loaded into the wells: the safety cover was placed onto the electrophoresis chamber. Finally, the leads were connected to the power source, allowing electrophoresis to begin. Moreover, after approximately 30 minutes, the DNA samples had migrated toward the positive (red) electrode. Thus, the gel was removed from the casting gel-tray and added to the viewing surface of the transilluminator. It is important to note, there should be no bubbles or trapped liquids under the gel, which could allow for potentially skewed visuals. Regardless, the results of the gel were observed and visualized, as the transilluminator revealed the DNA bands. Lastly, the surface of the transilluminator was cleaned and the gel was disposed of properly.

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