Report 1
Analysis of A. fischeri chDNA restriction digestion by Sal I enzyme after isolation and purification via agarose gel electrophoresis
Bio 219-064 (Techniques in Molecular Biology)
Group 5: Tung Nguyen, Uyen Tran, Amber Beckley, Danielle Exler
Department of Biology, Drexel University, Philadelphia, Pennsylvania 19104
Submitted October 27, 2014
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Abstract Bioluminescence is one of the benefits that a deep sea organism received from maintaining a symbiotic relationship with bioluminescent bacteria. Bioluminescence is controlled by the lux operon expression inside the bacterial cell. The lux operon, approximately 8.5 Kb in length, is composed of luxR and luxICDABE. The ultimate goal of this experiment is to create a restriction map of the Aliivibrio fischeri (A. fischeri) lux operon from the re-isolated plasmids after transforming the E. coli with the plasmid library. There are multiple steps for this experiment starting with the isolation of A. fischeri chDNA. Using Qiagen DNeasy Mini Spin Column Kit, three different samples of 100 uL of isolated A. fischeri chDNA solution each were obtained. Next, to determine the purity and concentration of the isolated A. fischeri chDNA samples, UV spectrophotometry was performed using two different spectrophotometers. The best sample was chosen for the next step in the experiment, restriction digestion, which had a concentration of 196.6 ug/uL. Purity analysis shows a 1.83 for the ratio of A260/A280 and a 2.15 for the ratio of A260/A230. Both of these values are within the normal range of the literature value of these ratios, which is an indication that this sample is pure and free of contaminants. Restriction digestion of three pure A. fischeri chDNA samples was performed using Streptomyces albus G (Sal I) restriction enzyme. Restriction digestion samples were analyzed by agarose gel electrophoresis. From the gel image, the digested A. fischeri chDNA
Bio 219-064 (Techniques in Molecular Biology)
Group 5: Tung Nguyen, Uyen Tran, Amber Beckley, Danielle Exler
Department of Biology, Drexel University, Philadelphia, Pennsylvania 19104
Submitted October 27, 2014
________________________________________________________________________
Abstract Bioluminescence is one of the benefits that a deep sea organism received from maintaining a symbiotic relationship with bioluminescent bacteria. Bioluminescence is controlled by the lux operon expression inside the bacterial cell. The lux operon, approximately 8.5 Kb in length, is composed of luxR and luxICDABE. The ultimate goal of this experiment is to create a restriction map of the Aliivibrio fischeri (A. fischeri) lux operon from the re-isolated plasmids after transforming the E. coli with the plasmid library. There are multiple steps for this experiment starting with the isolation of A. fischeri chDNA. Using Qiagen DNeasy Mini Spin Column Kit, three different samples of 100 uL of isolated A. fischeri chDNA solution each were obtained. Next, to determine the purity and concentration of the isolated A. fischeri chDNA samples, UV spectrophotometry was performed using two different spectrophotometers. The best sample was chosen for the next step in the experiment, restriction digestion, which had a concentration of 196.6 ug/uL. Purity analysis shows a 1.83 for the ratio of A260/A280 and a 2.15 for the ratio of A260/A230. Both of these values are within the normal range of the literature value of these ratios, which is an indication that this sample is pure and free of contaminants. Restriction digestion of three pure A. fischeri chDNA samples was performed using Streptomyces albus G (Sal I) restriction enzyme. Restriction digestion samples were analyzed by agarose gel electrophoresis. From the gel image, the digested A. fischeri chDNA
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