Dna Restriction Enzyme Lab

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Restriction enzymes are enzymes that can be used to cut DNA fragments into small fragments and they can recognize a very specific sequence of DNA (Lab Handout). In this lab activity, the restriction enzymes that we used were EcoRl, BamHl, and HindIII. The EcoRl enzyme cuts DNA sequences at the top strand 5’-GAATTC-3’ after G and the bottom strand 3’-CTTAAG-5’after A, BamH1 cuts DNA sequences at the top strand 5’-GGATCC-3’after G and the bottom strand 3’-CCTAGG-5’after G, and HindIII cuts DNA sequences at the top strand 5’-AAGCTT-3’ after A and the bottom strand 3’-TTCGAA-5’after A.
In this activity, we used gel electrophoresis to separate DNA fragments based on their sizes as the DNA fragments migrate from the negative to the positive pole. …show more content…
My lab partners were Megan and Kanthicha. We prepared the materials along with our instructor that we would be use during the experiment. Then we placed the gel-casting tray over the center of the chamber and aligned the notches in the tray with the notches on the side of the electrophoresis chamber. We inserted the heavy metal dams at the end of the gel-casting tray and pushed down firmly to be sure the seal was maintained. After that, we inserted the 8-well comb into the notches at the end of the gel-casting tray closest to the black end (negative pole) of the chamber. Then, we obtained one tube of 0.8 % agarose from the 65 degree water bath, which was liquid at that temperature. After that, we carefully poured the molten agarose into the gel-casting tray until the agarose almost reached the top of the tray, approximately 1 cm thick. Then we used a pipette tip to remove any large bubbles. It took about 15 minutes to let the agarose solidify as it cooled. Once the gel completely solidified, we carefully removed the metal dams at the ends of the gel casting-tray. We also removed the well comb from the agarose and pulled straight up. After that, we placed the electrophoresis chamber in close proximity to the power supply. The gel was ready to load with DNA samples after we filled the electrophoresis chamber with TAE (tris acetate EDTA) electrophoresis running buffer. (Lab …show more content…
Then we set the 20-200 micropipetter to 30μl. We picked up a fresh pipette tip for each sample using the pipette. Then we depressed the plunger with our thumb to the first stop position. After that, we placed the end of the pipette tip in the bottom of the sample tube and we slowly released the plunger to fill the pipette tip for 30μl of practice dye loading solution. We placed both elbows on the lab bench and held the micropipette over the gel with both hands. Then we gently lowered the tip into one of the wells. We released the pipette tip and replaced it with a fresh one. After loading the 4 practice wells, we took turns and we loaded our DNA samples into the appropriate wells. Then we gently pipetted the sample up and down to be sure it is thoroughly mixed. After that, we loaded 30μl of each DNA sample above and loaded it into a well. We used a separate pipette tip for each sample to avoid cross contamination. (Lab Handout)
Overall, we closed the top of the electrophoresis chamber and connected the electrodes (black to black and red to red). We plugged the power supply into an electrical outlet and turned on the power supply. We set the power supply to run at 100 V constant voltage. Then we pushed the RUN button to start the electrophoresis. We saw bubbles rising in the buffer and the loading dye moved from the well towards the positive end of the gel. After that, we allowed the electrophoresis to
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