Lab5_Wilkerson - Christopher Wilkerson 1 PCR Analysis and Agarose Gel Electrophoresis Objective The purpose of this lab was to analyze the DNA

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Figure 1. PCR reaction with steps illustrated. Figure 2. Electrophoresis diagram with sample run gel on left horesis with only right eight wells filled. Right most well contains 10 kb DNA ladder. oresis gel. All twelve wells were used and 10 kb DNA ladder was on the far left Here is a 10 kb DNA ladder with base lengths marked for reference. Christopher Wilkerson 1 05:44:28 PCR Analysis and Agarose Gel Electrophoresis Objective : The purpose of this lab was to analyze the DNA preps that were made previously to determine whether or not the plasmid that was meant to b purified was indeed obtained. PCR stands for polymerase chain reaction, and is a widely used method for amplifying DNA or RNA segments (genes). The PCR reaction consists of three parts: Denaturation, Primer Annealing, and Extension. Denaturation is the process of the individual DNA strands separating from the double helix structure. The PCR mixture is heated to 94 ° C, which provides enough energy for the hydrogen bonds between the nitrogenous bases to break. This allows each anti-parallel stand to be free for annealing. Next, during the Primer Annealing stage, the solution is cooled to 62 ° C. This is a point in which the specific primers in solution can begin to from hydrogen bonds at the beginning of the gene of interest. Two separate primer types are in the solution. Each attaches to a complementary strand of DNA at the gene locus. The third step of PCR is extension. This is the method of dNTP (nucleotides) in solution adding on to the primer in the 5’ to 3’ direction complementary to the template strand and creating the second strand of DNA, effectively doubling the amount of genetic material. DNA polymerase is required to perform this operation. The specific enzyme that we will use is known as TAQ ( Thermus Aquaticus ) DNA polymerase. This was derived from the marine species for which it is named that thrives on thermal vents in the ocean. TAQ polymerase operates most effectively at 72
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This note was uploaded on 06/23/2008 for the course CH 204 taught by Professor Leytner during the Spring '08 term at University of Texas at Austin.

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Lab5_Wilkerson - Christopher Wilkerson 1 PCR Analysis and Agarose Gel Electrophoresis Objective The purpose of this lab was to analyze the DNA

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