DNA lab report - Orett White Jordan Trecki Analysis and...

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1 Orett White Jordan Trecki 11-27-2008 Analysis and extraction of DNA from E. coli Introduction: Subject: the main goal of this experiment was to successfully isolate, extract and extract chromosomal DNA from E-coli cells and then analyze and check the DNA’s identify the usage Dnase and electrophoresis. The question to be answered was exactly how pure was the extracted chromosomal DNA? Hypothesis: In most sample used there is a larger percentage of RNA than DNA present. Given this fact it is possible that some contamination took place during the extraction phase of the experiment. This contamination could have resulted in some impurities in the final DNA sample. Method: The method was in variety of steps. The sample was first denatured, treated with a variety of substance for comparison. The samples were then stained using ethidium bromine and then placed in agarose gel. An electrical current was then run through the gel to separate and analyze the nucleic acids Materials and Methods: See attached protocol for more information. Lanes seven and eight were switched.
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2 Results:
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3 Discussion : DNA is the building block of all life. It is present in all organisms on the planet. DNA is important because it give the organism the layout for all proteins in the body. During the lab the purpose of the experiment was to extract chromosomal DNA from E-coli bacteria and then analyze the extracted DNA by using agarose gel electrophoresis. I hypothesized that since RNA is present in a cell much higher concentrations than DNA it’s possible that some contamination may have occurred during extraction phase of the experiment resulting in some impurities in the final sample. This hypothesis was brought about because with the equipment available to us and the large concentrations of RNA present with the DNA, it would be almost impossible to not have some impurities in our extracted sample. In order to extract the DNA we obtained a bacterial suspension of E-Coli in saline EDTA, and treated it with various amounts of chemicals and then centrifuged this mixture in order to create a mixture in which DNA was located in the top aqueous layer. We extracted this layer and treated it with 95% ethanol in order to allow the DNA top precipitate from the aqueous layer. We then treated the DNA in various percentages of ethanol in order to further purify it. We then loosened the DNA by placing it in STE and then placed it in a microfuge tube and stored it in a freezer for a week in order to prepare it for analysis. In the next part of our experiment we used our extracted DNA and used it to created 6 preps with various amounts of DNase, water and buffer all at various temperatures, as well as creating two preps of tRNA. Each prep was immersed in a solution of ethidium bromide (a mutagen) which diffuses into the gel and tightly binds to nucleic acid. We then
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4 conducted agarose gel electrophoresis which separated the nucleic acid and moved the nucleic acids through the gel, with smaller molecules moving the farthest. This
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