Identification of Unknown Plasmid
I. Title
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.
II. Abstract
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 containing antibiotics, we were able to examine the recombinant DNA of the bacteria. After incubation of the plates, we analyzed the samples and found that our unknown plasmid reacted positively on the LB/AMP plate. There were a total growth of three colonies on the LB/AMP plate and a negative result on the LB/KAN plate. With this data along with the positive reaction of pAMP on the LB/AMP plate, we came to the conclusion that our unknown plasmid was pAMP. In our next experiment, we analyzed the DNA via gel electrophoresis. First, we had to treat our unknown plasmid. Three treatments were performed: Uncut (U), single cut (S) with HindIII, and double cut (D) with HindIII and Bam H1. The gel was then stained with Ethidium Bromide, often used in chromatography, in order for us to view the gel under UV light. A photograph of the result was then printed out. This allowed us to determine the migration of each sample along with the number of base pairs in each fragment. Standard fragments of DNA were used to determine the size of our unknown plasmid, which at this point was pAMP. With the use of both pKAN and pAMP plasmid maps, we were able to solidify our conclusion that the unknown plasmid was pAMP.
III. Introduction The
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.
II. Abstract
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 containing antibiotics, we were able to examine the recombinant DNA of the bacteria. After incubation of the plates, we analyzed the samples and found that our unknown plasmid reacted positively on the LB/AMP plate. There were a total growth of three colonies on the LB/AMP plate and a negative result on the LB/KAN plate. With this data along with the positive reaction of pAMP on the LB/AMP plate, we came to the conclusion that our unknown plasmid was pAMP. In our next experiment, we analyzed the DNA via gel electrophoresis. First, we had to treat our unknown plasmid. Three treatments were performed: Uncut (U), single cut (S) with HindIII, and double cut (D) with HindIII and Bam H1. The gel was then stained with Ethidium Bromide, often used in chromatography, in order for us to view the gel under UV light. A photograph of the result was then printed out. This allowed us to determine the migration of each sample along with the number of base pairs in each fragment. Standard fragments of DNA were used to determine the size of our unknown plasmid, which at this point was pAMP. With the use of both pKAN and pAMP plasmid maps, we were able to solidify our conclusion that the unknown plasmid was pAMP.
III. Introduction The
Cited: 1.) Micklos, D., and Freyer, G. 1990. DNA Science: A first course in recombinant DNA technology. Cold Spring Harbor Press, New York, 477 pages 2.) Bennethum, T. M., J. A. Chiscon, M. O. Chiscon, C. R. Carlin, R. H. Shippee, and J. W. Vanable. 1993. Identification of an unknown plasmid. Pages 129-138, in Laboratory manual for Biology 225-6: The Basic Concepts. Department of Biological Sciences, Purdue University. 3.) Lawrence, S.M., M.K. Heidemann, and D.O. Straney. 1998. Biological Science111L Laboratory Manuel, 2nd edition. P. 60. Hayden-McNeil, Plymouth