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This 5 page Class Notes was uploaded by Alfonso Reynolds on Wednesday October 28, 2015. The Class Notes belongs to CHEM 1050L at Weber State University taught by Kyle Ashby in Fall. Since its upload, it has received 18 views. For similar materials see /class/230795/chem-1050l-weber-state-university in Chemistry at Weber State University.
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Date Created: 10/28/15
Unknown Plasmid DNA Fragment Data Table B Cut by Different Enzymes Unknown Plasmid Distances Migrated mm Observed Fragment Sizes Base Pairs A00 1 95 3450 Apa I 91 3900 BglI 12133 16501150 Eco RV 95 3450 Pvu II 105 2550 an I 95 3450 Uncut Control 113 2060 Since the late 1970 s scientists have used agarose gel electrophoresis as a technique to separate fragments of DNA The electric current introduced in the agarose gel causes the negatively charged DNA fragments to move through the gel towards the positive cathode the smaller fragments move farther than the larger ones The agarose gel is often stained with ethidium bromide to make the DNA fragments Visible under ultraviolet light In this experiment plasmid DNA was put through restriction digestions with different restriction enzymes and run through gel electrophoresis in order to determine the identity of a mystery plasmid labeled A Eight reaction tubes were prepared for gel electrophoresis Six contained plasmid DNA and each with a different restriction enzyme one contained the uncut plasmid DNA which served as the control and one contained lambda DNA cut with the Hind III enzyme which served as the ladder The control more specifically the negative control was used to determine the number of base pairs in the entire mystery plasmid DNA since it was not cut by any enzyme The ladder or molecular weight standard was used to construct a standard curve using known base pair lengths The data used to construct this standard curve was recorded in Table A shown below and the standard curve was drawn in Figure B shown later Lambda DNA was used as the size standard for several reasons One is that it is very easy to obtain and thus cost effective More importantly however the entire 48000 base pair sequence is known Lambda DNA Cut with Hind HI Enzyme Table A to Construct Standard Curve Distance Traveled mm Fragment Size Base Pairs 4 23130 55 9416 64 6557 79 4361 105 2322 1 1 1 2027 The two smallest DNA fragments 546 and 125 base pairs were not visible on the gel electrophoresis photograph The other siX fragments were matched with their respective distances traveled and used to plot points on the graph for the standard curve How do restriction enzymes work and how can these restriction enzymes be indentified According to Dr Geoffrey G Wilson s study of restriction and modification systems of DNA along with countless other similar studies by scientists restriction endonucleases cleave double stranded DNA into fragments at specific points which are then broken down further by other endonucleases This prevents infection by destroying the foreign DNA or virus in the DNA strand introduced by the bacteriophage Wilson A few weeks ago our lab used gel electrophoresis to analyze the digested lambda DNA and the restriction enzymes used to break down the lambda DNA The enzymes used were Eco RI Bam HI and Hind 111 After performing this test the gel electrophoresis photograph was analyzed and plotted onto a semi log graph paper The first lane in the photograph contains a ladder used to tell the size of the other samples and the other columns or lanes show different sizes of DNA in the other samples When plotting the distance traveled in the photograph we first labeled the y aXis on our graph as the base pairs and the X aXis as the distance that the lambda DNA cut with Hind 111 had migrated Next we used the photograph and the ladder in the photograph to measure how far each DNA fragment had traveled using millimeters The following list of the amount of base pairs shows how far each must travel according to the photograph 23130 base pairs10mm 9416 base pairsl4 mm 6557 base pairs l6mm 4361 base pairs25mm 2027 base pairs27 mm and 564 base pairs42mm Using these numbers I was able to then draw a standard curve line on the graph A control labeled D was used to show the process of the DNA under gel electrophoresis without any enzyme Next our group was told to find the mystery plasmid Since we had already created a semi log graph we were able to determine which two lanes in the photograph were noncutters or samples that had not moved These noncutters looked like a solid line with no other lines migrating away from them The two lines that had not moved were measured and then using the graph we determined that the two noncutters were Apa I and EcoR V According to our lab manual the plasmid containing these two noncutters is pUCl l9 and this is our mystery plasmid Using such tests can not only help students at UGA discover their mystery plasmid but DNA restriction analysis has many bene ts in the real world as well Doctors and scientists can use DNA restriction analysis to discover an unknown bacterium or virus This process is known as Single Genome Fingerprinting and after identifying the restriction sites in the virus the doctors can use to the test to allow rapid identi cation of microorganisms with applications in forensics medicine public health and environmental microbiology Ferris Matthew M et al This identi cation can be bene cial in identifying a mysterious bacterium or virus that is plaguing a small community or even a culture such as the E Coli breakout in Atlanta s waterpark Whitewater Furthermore restriction analysis can not only identify bacteria but can also identify handicaps or impairments in humans such as the restriction site found in the 128 rRNA gene used to detect non syndromic hearing losss in Tunisian patients MkaouarRebai Emna et al These are just a few examples of how enzyme restriction analysis can be bene cial to health and science Ibelieve that discovering the diseases that harm so many people is very bene cial in trying to eradicate future harm to people such as discovering E Coli in fruit that might be carried at local grocery stores I do not believe however that discovering a disease is any more important than identifying restriction sites in genes of patients with disorders or visa versa Works Cited Ferris Matthew M et al quotFingerprinting of single viral genomesquot Analytical Biochemistry 3372 15 Feb 2005 278288 MEDLINE with Full Text EBSCO Library name City State abbreviation 9 Apr 2008 lthttpsearchebscohostcomloginaspxdirectrueampdbmnhampANl56915 08ampsiteehost livegt MkaouarRebai Emna et al quotNew polymorphic mtDNA restriction site in the 12S rRNA gene detected in Tunisian patients with nonsyndromic hearing lossquot Biochemical And Biophysical Research Communications 3693 09 May 2008 849852 MEDLINE with Full Text EBSCO Library name City State abbreviation 9 Apr 2008 lthttpsearchebscohostcomloginaspxdirect4rueampdbmnhampANl83253 29ampsiteehost livegt Wilson G G and N E Murray quotRestriction and modi cation systemsquot Annual Review Of Genetics 25 1991 585627 MEDLINE with Full Text EBSCO Library name City State abbreviation 9 Apr 2008 lthttp searchebscohostcom lo gin aspxdirectrueampdbmnhampAN l 8 l 28 l 6ampsiteehost livegt
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