Identification of an Unknown Plasmid
In this experiment, we determined the phenotypic capability of an unknown plasmid along with its size. With the use of gel electrophoresis, we analyzed the gel photograph by using a standard DNA marker, Lambda HindIII, and came to a conclusion based on our results.
Two experiments were done to identify an unknown plasmid. The success of these experiments came from the use of modern day technology involving gel electrophoresis. First, bacterial transformation to E. Coli DH5 was performed on our unknown plasmid along with two known plasmids, pAMP and pKAN, and a negative control TE, a buffer without DNA. By performing confluency streaking of bacteria in plates
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The other two bacterial mechanisms, conjugation and transduction, are options with certain circumstances, but can be inconsistent under normal conditions. Because of this, transformation is the ideal and most commonly used mechanism when transferring naked DNA. In our first experiment we plated three samples of DNA, pAMP, pKAN, and an unknown, along with a TE buffer without DNA onto the LB, LB/AMP and LB/KAN plates. We hypothesized that a positive growth of our unknown on LB/AMP would mean that our unknown was pAMP, but if the growth were on the LB/KAN then our unknown was pKAN. After transformation is done, phenotypic changes can be viewed by way of gel electrophoresis. Phosphate groups making up the backbone of the double helix in DNA contain negatively charged oxygen, making electrophoresis a primary way of sorting the DNA by size. Restriction enzymes, HindIII and Bam H1 were treated to our unknown DNA. Three treatments were done to better analyze our samples: Uncut, single cut, and double cut. This was a necessary procedure because it helped to determine the sizes of the cut and uncut plasmid samples compared to a standard lambda marker. The samples of DNA were then placed in a well containing 0.8% agarose gel, which is ideal for efficiently separating fragments. After the DNA