BME103 s2013:T900 Group5
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Lab Write-Up 1
Lab Write-Up 2
Lab Write-Up 3
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LAB 1 WRITE-UP
Initial Machine Testing
The Original Design
The PCR machine, also known as a thermocycler, is a device used in laboratory settings to raise and lower the temperature of various segments of DNA. Through this process the samples of DNA can be amplified using the polymerase chain reaction to create several copies of the DNA sequence. This technique of raising and lowering temperature to initiate the polymerase chain reaction is crucial to modern medicine as it allows the cloning of vast amounts of DNA to be utilized for other means such as DNA sequencing.
Experimenting With the Connections
When we unplugged (part 3) from (part 6), the machine ... (did what? fill in your answer)
When we unplugged the white wire that connects (part 6) to (part 2), the machine ... (did what? fill in your answer)
(Write the date you first tested Open PCR and your experience(s) with the machine)
The Experimental Protocol Planner developed and carried out the methods of a Polymerase Chain Reaction protocol.
DNA Sample Set-up
+: Positive Control
DNA Sample Set-up Procedure
1.) Use the micro-pipet to dispense 50 μL of PCR reaction mix into 8 vials, be sure to change the transfer pipettes to avoid cross contamination.
PCR Reaction Mix
DNA/ primer mix
Research and Development
Specific Cancer Marker Detection - The Underlying Technology
Polymerase Chain Reaction, or PCR, is the occurrence in which a segment of DNA is replicated multiple times. For the reaction that we are trying to produce the solution we will be using will contain Template DNA, Primers, Taq Polymerase, Magnesium Chloride and dNTP's.
To begin the process of PCR the solution containing template DNA we take the template DNA and heat up the solution to 95°C for 30 seconds in order to break the hydrogen bonds between the nucleotides. This exposes the bases to allow the primers, which are short sequences of DNA that have been artificially synthesized, to attach to the exposed bases on the template DNA. In this process there are forward primers and reverse primers that attach to the exposed template DNA. After the primers are added, the solution is cooled down to 57°C in order to initiate primer annealing, or binding, to their respective regions. To complete a full strand of DNA the solution must be raised up to 72°C. The raise in temperature activates the Taq polymerase. Taq Polymerase is a protein that allows for binding of dNTP which are individual bases floating around within the solution that act as the subunits that the polymerase use to build new strands. Within the solution the dNTP's bind to the exposed bases starting from the primers. To aid in this process, the added Magnesium Chloride acts as a cofactor to allow polymerase to work and aids with the binding process. Taq polymerase will seek out and react with double stranded parts of DNA and can bind after it attracts individual bases which attach to the primer through a covalent bond, thus creating a new strand of DNA. In a PCR reaction this entire process is repeated multiple times, preferably 30 in order to cancel out other genes and work just for genes that code for cancer.
The reason that the process only codes for the cancerous genes is that the primers were artificially synthesized in order to code for those genes. For the non-cancer gene there will be no binding of the primer since it does not match the same sequence of DNA as the cancer gene. As a result the non-cancerous gene will remain uncoded while the cancerous gene is continuously copied which allows for the development of an new strand by the Taq polymerase and the dNTP's within the solution.