Molecular Biology Lab Report Essay

1659 WordsApr 7, 20117 Pages
Molecular Biology Lab Report Mapping DNA using Restriction Enzymes Ava II and Pvu II to cut Bacterial DNA Abstract The objective of this project is to map bacterial DNA, which is derived from E. coli, using restriction endonucleases with gel electrophoresis. The DNA fragments, after cutting has occurred, are separated using agarose gel electrophoresis. The DNA fragments are placed in the gel, and an electric current is run through the matrix of the gel-like agarose. Migration of the fragments across the gel is based on the size and charge of the fragment. After the fragments have been run in the gel, they are stained with methylene blue and viewed on a light box. This allows the DNA bands to be viewed on the gel. Introduction This…show more content…
2. Prepare the digestions a. Three color-coded microtubules holding deionized water and an appropriate buffer were already prepared by the instructor. The three microtubules were labeled A (for Ava II), P (for Pvu II), and AP (for both). The color of each microtubule was recorded. b. 2 µL of pUC 19 DNA solution was added to each tube. The bottom of each tube was tapped on the laboratory bench in order to facilitate the movement of DNA to the bottom of the test tube. c. The appropriate enzymes were added to each test tube. The bottom of the test tube was tapped on the laboratory bench to make sure that the reaction mix is at the bottom of the test tube. d. The test tubes were placed in a water bath at 37o C for at least 45 minutes to an hour. 3. Prepare and pour the agarose gel The instructor made the gel using the following procedures: a. 100mL of 1X Tris-borate-EDTA, or 10X TBE was added to 0.8g of agarose in a 250-mL Erlenmeyer flask. b. The flask was covered with a plastic wrap and placed on a hot plate until it boiled. The solution was swirled sporadically. One had to wear protective gloves while stirring the solution. The solution was allowed to boil until it is clear, showing that the agarose was in the solution. c. The solution was allowed to cool to approximately 50o C. The cooling process could be hastened by cautiously whirling the flask under

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