An Experiment For The Unknown Plasmid

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This experiment was very successful as a credible restriction map for the unknown plasmid could be constructed. Within this experiment, both single digest and double digests consisting of three restriction endonucleases were used in order to map out the restriction sites of the enzymes making up an unknown plasmid. In order to separate the DNA fragments by their distinct number of base pairs, it was necessary to run an agarose gel electrophoresis. Within the gel electrophoresis, it is necessary to run a 1kB ladder in the first well. This ladder contains numerous known lengths of base pairs, and is run next to and unknown product in order to approximate the sizes of unknown fragments simply by comparing the unknown fragments to the coinciding fragments of the known ladder. This ladder gives us the ability to precisely and accurately draw conclusions about the results derived from the gel electrophoresis as it serves as an essential reference point. Because of the known fragments in the ladder, we were able to create a standard curve. Within the standard curve, the distance the fragments traveled was plotted against the length of the known base pairs within the ladder. Once the points were plotted, a line of best fit was constructed and an equation of the line was electronically derived. By plugging in the measured distance of how far the fragments traveled, shown by “x”, into the equation for the line of best fit, the lengths of the base pairs created by the restriction enzymes was able to be calculated.
In order to make the final step of mapping out the restriction sites on an unknown plasmid, it was essential to first map out the single digest maps. The single digest maps were created using both the number of fragments produced...

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...lasmid have the capability to survive, and multiple in number as they expand and reproduce. In addition, restriction enzymes have led to gateway discoveries in the topic of cloning. Essentially, because these restriction enzymes have allowed for the removal of a fragment of DNA and for it to be placed in another location, this idea has led to scientists being able to integrate exogenous DNA into natural plasmids that may ultimately lead to cloning plasmid vectors. These plasmids then have the ability to self-replicate (neb.com). The discoveries made surrounding these restriction enzymes have paved the way for the cloning of DNA. Furthermore, DNA mapping is a practical application stemming from restriction analysis that now allows for scientists to be able to detect insertions and deletions, single nucleotide polymorphisms, and identifying genetic disorders (neb.com)

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