BME100 f2014:Group12 L4
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OUR TEAMLAB 4 WRITE-UPProtocolMaterials
1. The qualitative data in this lab is the color of the samples. The quantitative data is the volume measurements of the samples in μl. 2. Accuracy reflects how close a measurement is to an accepted value while precision reflects how close measurements are to each other. 3. {need results}
OpenPCR program
Denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds, and Extend at 72°C for 30 seconds
Research and DevelopmentPCR - The Underlying Technology
During a PCR reaction, the function of the template DNA is to provide the pattern for the sequence of nucleotides in an RNA transcript. Each gene has two DNA strands that make up that particular gene. The function of the primers during the reaction is to attach to specific sites on the DNA strands on either end of the segment that will be copied. Before the unraveled DNA strands can pair up, the primers attach to the specific part of the segment, in order to be able to replicate the DNA. During a PCR reaction, the Taq polymerase locates the primer which is attached to a single strand of DNA. After locating a primer, the Taq polymerase then begins to add complementary nucloetides on the strand until the end of the strand in order to complete the duplicated DNA strand. The deoxyribonucleotides (dNT's) are the single units of DNA. Each deoxyribonucleotide includes a nitrogenous base, a deoxyribose sugar,and one phosphate group. During the PCR reaction, deoxyribonucleotides will polymerize to form DNA. The phosphate group from one nucleotide will bond to the 3' carbon on another nucleotide, forming a bond within the DNA.
During the initial step in the PCR reaction, the thermal cycler heats to 95 degrees Celsius for 3 minutes. During the 3 minutes the Template DNA, primers, Taq polymerase, and the deoxyribonucleotides (dNT's) all heat up in order for the DNA to be able to separate. In Denature, the temperature stays at 95 degrees Celsius and the double stranded DNA separates into two strands. After the DNA is separated into two single strands, the thermal cycler cools down to 57 degrees Celsius for 30 seconds. In this step, Anneal, the primers then attach to complementary matches on the target DNA sequence, in order to be ready to replicate. Then the thermal cycler heats up to 72 degree Celsius for 30 seconds in the Extend step. During this step, the Taq polymerase attaches to the primed sequences, and then adds nucleotides in order to extend the second stand of the DNA. This then completes the first cycle. These steps are repeated in subsequent cycles in order to create more DNA copies. After the 35 steps of the PCR reaction are completed, the mixture is held at a constant 72 degrees Celsius for 3 minutes during the final step in order to complete the extension process of the DNA. Finally, the mixture is held at 4 degrees Celsius during the final hold until it is removed from the thermal cycler and refrigerated for further use.
The base Adenine (A) pairs with the base Thymine (T). The base Cytosine (C) pairs with the base Guanine (G).
During the PCR reaction, base pairing occurs during both the anneal and extend steps. In the anneal step, there is a specific sequence of nucleotides that are found within the primer sequences that are connected to their corresponding sequences in the template DNA. The rules of base pairing, A pairs with T, and C pairs with G, allow for this to occur in the anneal step. In the extension step, the Taq polymerase is used to pair the deoxyribonucleotides to their corresponding base on the template DNA in order to complete the sequence. During both of these steps, base pairing occurs in order to complete the copied DNA sequence.
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