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Gel Electrophoresis Synthesis Lab Report

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The purpose of this experiment was to digest plasmid DNA and analyze it by agarose gel electrophoresis. The plasmid pBR322 was introduced to two restriction endonucleases, Hind III and Hinc II. These restriction endonucleases are meant to cleave a part or parts of the sequence of the plasmid DNA; in this case Hind III should cleave one part of the sequence while Hinc II cleaves at two parts. To observe this cleavage two reaction mixtures were created, one with Hind III and the other with Hinc II, along with other reagents that were necessary for the mixture. These mixtures were heated at 37°C for 90 minutes. While these mixtures were heated a 5 X TBE buffer was created. This 5 X TBE buffer contributed to the creation of the 1% agarose gel and the 0.5 X TBE buffer that were going to be used during the gel electrophoresis process. …show more content…

Once the loading solutions were made and the agarose gel electrophoresis apparatus was set up, the solutions were dispensed into the wells of the gel, as well as a reference solution. The gel image gathered from the gel electrophoresis allowed for the analysis of the plasmid DNA and the effects the restriction endonucleases, Hind III and Hinc II, had on it. While analyzing the gel image, it was apparent that based on the reference solution in lane 1 and the mark in lane 3, the Hind III solution, that the number of base pairs should be the same. Also it was apparent that lane 5, the Hinc II solution, had 4 marks. By measuring the migration distance in centimeters and plotting against the log(# of base pairs(bp)) for the standard in lane 1 the linear trendline equation provided by the calibration curve allows for the number of base pairs to be found for the marks in lane 3 (Hind III) and lane 5 (Hinc II). After a series of calculations, the number of base pairs for each mark in lanes 3 and 5 were

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