Using DNA technology, including restriction enzymes, gel electrophoresis, and transformation we performed two experiments to identify an unknown plasmid. The first experiment involved gel electrophoresis of plasmid DNA with and with out restriction enzymes to determine migration and number of base pairs in specific fragments. Known DNA fragments were used to determine the size of the unknown fragments. Plasmid maps of pKAN and pAMP were used to compare the sizes to the unknown sizes, where by identification of the unknown was pKAN. The second experiment used the same unknown plasmid in a transformation to bacteria E.coli HD . The recombinant DNA of the bacteria was analyzed by growing the bacteria in plates containing antibiotics. The growth of the unknown sample was shown only in the pKAN/LB plate and the LB (standard medium) plate, thus the bacteria contained DNA which held resistance to kanamycin, a characteristic of the pKAN plasmid. Through the results of both experiments we therefore conclude that the unknown plasmid was pKAN.

In Experiment 1 we observed the Standard DNA piece cut in several places with a restriction enzyme lambda Hind III, and we hypothesize that we would obtain several fragments upon Gel electrophoresis. The number of base pairs in each fragment was given and by measuring how far the pieces migrated we hypothesized that we could determine sizes of the unknown plasmid DNA pieces in relation to how far those pieces migrate. The second sample was U (uncut sample) of our unknown plasmid. No enzyme cut this sample, therefore we predicted there would be only one fragment shown in gel electrophoresis. This sample was in the supercoiled form and we predicted that it would migrate farther when compared to the S cut. The next sample was an S (single cut) sample of the same unknown plasmid. This sample of DNA was cut once with a restriction enzyme Hind III and therefore we predicted the shape would change to a linear fragment of DNA. The final sample was a D (double digested) sample of our unknown plasmid. This sample was cut with the restriction enzymes Band H1 and Hind III. Since the sample was cut twice, we hypothesize that it will show two DNA fragments. We further hypothesize that we will be able to determine the unknown plasmid based on the size of the fragments, as the enzymes Hind III and Bam H1 cut pKAN and pAMP at different places producing different size fragments. Using a plasmid map, we predict that we will identify the plasmid based on the data obtained from the gel electrophoresis.

In Experiment 2 we were given four microfuge tubes containing DNA and another tube of cells E. coli DH5. Of the four tubes, one was the unknown plasmid DNA from Experiment 1. The positive control was the DNA with known plasmid DNA in them. One positive control contained the DNA of pAMP and the other contained the DNA of pKAN. The negative control contained a buffer solution in the tube with no DNA present. After transformation was complete in the E.coli DH5, the cells were then cultured onto three different plates. One plate contained a standard growth medium (LB), the second contained LB and the antibiotic ampicillin, the third contained LB and the antibiotic kanamycin. We hypothesize that the bacteria which have the plasmids containing the gene for antibiotic resistance will be able to grow in the plates containing that antibiotic. We also predict that we can identify the unknown plasmid by which type of environment it grows in. We expected that the negative control would only grow in the standard medium since there was no DNA to transfer in that sample and no antibiotic resistance given.

Some common words found in the essay are:
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Approximate Pages = 15 (250 words per page double spaced)