Data Bases • Custom Term Papers • Free Term Papers • Free Research Papers • Free Essays • Free Book Reports • Plagiarism? Links • Top 100 Term Paper Sites • Top 25 Essay Sites • Top 50 Essay Sites • Search 97,000 Papers @ DirectEssays.com • Search 101,000 Papers @ ExampleEssays.com • Search 90,000 Papers @ MegaEssays.com Free Essays • Term Paper Sites • Chuck III's Free Essays • Free College Essays • TermPaperSites.com • My Term Papers • Get Free Essays • Essay World • Planet Papers	Search Lots of Essays Back to Subjects - Miscellaneous PCR PCR Using PCR and Gel Electrophoresis to Determine Genotype. Paul Phillips IV, Biology Department, Southwest Texas State University, San Marcos, TX 78666. In certain situations, it is necessary to identify DNA retreived from a sample. When there is a small sample in need of identification, Polymerase Chain Reactions are used to multiply the DNA in the sample in to many identical samples. The DNA retrieved from the reaction can then be imported into an aparatus using gel electrophoresis to compare the sample of DNA to other samples. In our experiment we learned the how to replicate tiny samples of DNA into usable amounts and how to analyze the specimine using gel electrophoresis. The samples of DNA were obtained by plucking individual hairs from students’ heads and using the PCR device to replicate the DNA from the roots of the hair. The replicated DNA samples were then placed into the electrophoresis gel and the device was turned on. Using the methods discussed above we found that three of the fourteen samples, 21%, were homozygous and the remaining eleven samples, 79%, were heterozygous. We concluded that it is possible to examine small amounts of DNA by first replicating the sample using Polymerase Chain Reactions then using gel electrophoresis to The main goal for our experiment was to learn how to examine DNA when there is only a small sample present. We examined the samples of DNA obtained from student hair using the replication method of Polymerase Chain Reaction and the inspection method of gel electrophoresis. The way the PCR method works is by first mixing a solution containing the DNA, DNA polymerase primers, and certain nucleotides. Next, the solution is heated to allow the strands to seperate, then cooled to allow the primers in the solution to bind to the strands. The last step occours when the DNA polymerase extends each 31 end of the primer (Campell, 1999). This process results in exact coppies of DNA molecules. The amplified samples of DNA were placed into the gel electrophoresis aparatus where we were able to see if the sample of DNA was heterozygous or homozygous. It is important for us to be able to replicate and examine DNA. In the event of violent crimes, small samples such as hair, skin, seamen, blood, or urine can give investigators a sort of genetic fingerprint of the criminal. The first step in employing the methods discussed above to replicate and investigate DNA was to obtain a sample. Our samples were obtained by plucking hair from students in our class. The root of the hair was isolated and placed into a small microfuge tube. The tube with the hair root was then incubated in 100 ul of digestion buffer containing 6ug of proteinase K for one hour at 55°C then for ten minuets at 95°C using the thermocycler in the lab room. When the DNA extraction was cooled, the tubes were vortexed for thirty seconds. After that a new microfuge tube was set up by adding 15.0ul of the extracted DNA and 10.0ul of the reaction muxture. The reaction mixture contained 4.0ul of H2O, 2.50 ul of 10x PCR buffer without Mg, 1.25ul of DMSO and 20x dNTP’s, and 0.50ul of #1 and #2 primer. It should be noted that a hotstart PCR is required. This means that the Taq DNA polymerase is not to be added to the PCR reaction mixture until the solution has reached 95°C. The temperature conditions for the PCR are as follows: 1)five minuets at 95°C (add Taq DNA polymerase), 2)one minuet at 95°C, 3)one munuet at 65°C, 4)one minuet at 72°C. Upon conclusion of the fourth step, steps two through four are repeated twenty-nine more times. When thoes steps are finished, the temperature is held at 20°C. In step one, the hot start is initated by incubating the tubes for five minuets at 95°C and adding 0.4 ul Taq DNA polymerase to each tube while disallowing the tubes to cool and without taking time to mix the reaction solution after adding the Taq polymerase. When the PCR technique is completed, the tubes are stored at 4°C until analysis of the tubes. To alylize the PCR results with the gel electrophorese, 2.5ul of the 10x loading dye is added to each PCR reaction tube. The gel for the electrophorese consists of 1.5% agarose gel with 0.5x TBE and 200ng/ml ethidium bromide. The gels were run at 90-100 volts for 1-1.5 hours. Upon completion of the experiment we were able to examine the DNA. First, the electrophorese revealed that three of the fourteen samples were were homozygous while the other eleven were heterozygous. This conforms the fact that close to eighty percent of our population is heterozygous and twenty percent homozygous (Isprilus, 2000). Bibliography: none Word Count: 796
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