Isolating DNA - Polymerase Chains Reaction and Agarose Gel...

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Polymerase Chains Reaction and Agarose Gel Electrophoresis Name: Husing Vang Date: 2/1/2012 Course: BMB 442 Email: [email protected] Student ID: 9-1192-5333 Introduction: The overall goal of this experiment was to successfully amplify a portion of the pBR322 DNA sequence, and test the changes of concentration of template DNA with respect to the efficiency of the PCR reaction. Also, the results will be analyzed via an agarose gel electrophoresis to check for the amplifications molecular weight (MW) as well as the brightness of its bands. The process of amplifying DNA requires four essential materials: template DNA, DNA polymerase, nucleotides, and two synthetic primers. Also, a thermocycler is required to increase the temperature of the solution. In PCR, the first step to amplifying a DNA requires that all of the four essential materials are pipetted inside a special thin-walled PCR tube. This tube is special in that it has the ability to adapt quickly to the changes in temperature, which is essential in this procedure. Once all the materials are transferred into the PCR tube, the tube is then placed into a thermocycler where the
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PCR tube will undergo a series of changes in temperature. The use of temperature above 94 O C allows for the DNA to denature, thus gives the primers access to their respectful places. There are two types of primers: a downstream primer and an upstream primer. When DNA denatures, they split apart, leaving behind two single-stranded DNA, one in the 5’->3’ direction and another in the 3’ ->5’ direction. The downstream DNA is the 3’->5’ DNA strand, thus the downstream primer will form a complimentary bond to this strand. Also, the single-stranded DNA in the 5’->3’ direction is the upstream strand, therefore is complimentary to the upstream primer. Once the primers are complimentarily bonded (or annealed) to the respectful SS-DNA, the DNA polymerase will begin elongation until the section of the targeted DNA has been copied. In this experiment, we are using pBR322, a template DNA which is ready to undergo multiple rounds of PCR. Results and Calculations:
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This note was uploaded on 02/10/2012 for the course B M B 443w taught by Professor Staff during the Fall '08 term at Penn State.

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Isolating DNA - Polymerase Chains Reaction and Agarose Gel...

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