BME100 f2013:W1200 Group3 L4
 BME 100 Fall 2013
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Home People Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3 Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6 Course Logistics For Instructors Photos Wiki Editing Help | |||||||||||||
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OUR TEAM
LAB 1 WRITE-UPInitial Machine TestingThe Original Design
The OpenPCR is a technique used to copy DNA molecules. This processs is done by dramatic tempertaure changes to isolate DNA strands from one another. To begin the machine is heated to almost boiling to denature the two strands from the original DNA. Once the two strands are seperated, the temperature decreases to allow the primers to attach and bind to complimenterary matches of DNA. This initiates the coping process and the taq polmerase then attahces (slightly higher tempertaure) to add nucleotides to the sequence to extend the strand. The process is completed with the four strands of DNA. This is the first cycle and many more occur to have billions of strands duplicated. Experimenting With the Connections When we unplugged part 3 from part 6, the screen on the machine turned off.
After letting the machine run for about an hour, it was still on the first cycle. This was done on October 23,2013.
ProtocolsThermal Cycler Program
Research and DevelopmentPCR - The Underlying Technology (Add a write-up, essay-style, organized into paragrpahs with descriptive headers, based on the Q&A's from Section three of your worksheet) Components of Polymerase Chain Reaction: There are many components which all play a part in the process of Polymerase Chain Reactions. First, the original DNA strand is used as a template from which millions of copies are made during the process of PCR. Once the DNA sequence has been separated, the DNA primers will bind to both strands. These primers will bind to specific starting sequences or starting codons. Once both primers bind to the two template DNA strands, the Taq Polymerase bind to the end of the primer sequence. The Polymerases then add complementary nucleotides to the growing replicant strand until they reach a stop codon. Magnesium Chloride is needed in this step because Taq Polymerase is a Magnesium (Mg 2+)dependent enzyme. The Taq Polymerase is able to add nucleotides to the growing strand because dNTP's, a solution containing free-floating base pairs, is present in the solution. Polymerase Chain Reaction Procedure: The process of Polymerase Chain Reactions contains six main steps, three of which are repeated about 35 times. The first step is to prepare the PCR reaction device by keeping the heating block at 95 degrees Celsius for three minutes. After this step, the cyclic portion of the experiment begins. The template strand is heated to 95 degrees Celsius for 30 seconds. In the absence of DNA helicase to split the strands, instead this method supplies the strand with enough kinetic energy to split the double strand into the two complementary strands. The temperature of the reaction vessels is then lowered to 57 degrees Celsius for 30 seconds. This step, called annealing, is the optimal temperature in which the primers will attach to the split DNA strands. The annealing step sets up the extending step. In this step, the temperature is increased to 72 degrees Celsius. At this specific temperature, the Taq Polymerase enzymes add complementary nucleotides to the ends of the primer sequences. The denaturing, annealing, and extending procedures are repeated as necessary. On the last extending cycle, the temperature is left at 72 degrees Celsius for another three minutes. This ensures that the Taq Polymerase enzymes have enough time to finish adding complementary base pairs to all of the newly created DNA segments. Finally, the solution is held at 4 degrees Celsius in order to keep the solution at an appropriate temperature for short-term storage until other tests can be run on the newly amplified DNA.
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