BME100 s2014:T Group8 L4
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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
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LAB 1 WRITE-UP
Initial Machine Testing
The Original Design
The openPCR is an over-glorified oven. It's purpose is to heat the dna to specific temperatures very precisely in order to achieve PCR. The openPCR oven has a display that it uses for user-interface, and a usb port which is used to connect to the computer and change parameters. Other than that, the openPCR machine is a simple heating pad connected to a heatsink/fan, and the heatsink and fan are connected to an arduino. What the arduino does is that it takes the temperature from the heating pad, and depending on what the desired temperature is, it either makes the heating pad hotter, or to cool the heating pad, it speeds up the fan on the heatsink. In order for the arduino to do this, it has constant temperature monitoring.
When we unplugged the display screen (part 3) from the circuit board (Part 6), the machine's display turned off; this was a power cable for the display.
When we unplugged the white wire that connects the circuit board (Part 6) to the heating container (Part 2), the machine's display stopped showing a accurate temperature. This wire then, was the wire that told the arduino what the temperature was, and by association, told the display what the temperature was.
The Open PCR machine was first tested by our group on March 20, 2014. As a trial run we let the Open PCR machine run for about eight cycles, which took approximately thirty minutes to complete. The specific machine that our group used complied with the partnered computer software and our custom heating/cooling protocol, and the trial run ran without errors or significant delays and thus proved the machine was completely functional and therefore passed. There were also no visible problems on the inside when we opened it up nor on the outside/heating pad.
Thermal Cycler Program
DNA Sample Set-up Procedure
1. Obtain 8 empty PCR tubes, tube-holding rack, positie control DNA, negative control DNA, patient DNA (3 from each patient) and a pipette with disposable tips.
DNA/ primer mix
Research and Development
===PCR - The Underlying Technology===
Functions of PCR Components
Each PCR reaction starts with the template DNA. These function as the master copy for the gene to be replicated. The primers attach to opposite ends of the two strands once the DNA has been denatured, and serve as an attachment point for the Taq Polymerase. Taq Polymerase, an enzyme important to PCR because of the high temperature, travels along the template DNA--and later the new DNA copies--adding nucleotides to the primer to replicate the DNA. THe deoxyribonucleotides are DNA-building blocks that make up the Code that DNA is comprised of.
Deoxyribonucleotides in DNA and ribonuculeotides in RNA are the components of the biological code that makes up genes. There are four nucleotides for DNA: Adenine (A), Thymine(T), Cytosine (C), Guanine (G). When combined into dual-stranded DNA, Adenine bonds with Thymine and Cytosine bonds with Guanine.
Thermal Cycling makes PCR possible. Once all components have been placed in the PCR tubes and the machine started, it heats up to 95°C for three minutes to get up to temperature and start to unravel the DNA double strand. The 95°C is maintained for another 30 second during the Denaturing Phase to separate the two strands on DNA. Then, the temperature drops to 57°C for 30 seconds in the Annealing Phase so that the primers can attach at the desired codon for the target gene of replication. The next 30 seconds are spent at 72°C for 30 seconds in the Extending Phase, where Taq Polymerase attaches to the end of the primer and begins copying DNA. In the Final step, maintaing 72°C for 3 more minutes, Taq Polymerase finishes relication. The Final Hold at 4°C allows all genes and components to settle and maintain their structure.