BME103:T130 Group 13
BME 103 Fall 2012 | Home People Lab Write-Up 1 Lab Write-Up 2 Lab Write-Up 3 Course Logistics For Instructors Photos Wiki Editing Help | |||||
OUR TEAMLAB 1 WRITE-UP
When we unplugged the PCB Board of the LCD from the Open PCR Circuit Board, the machine's LCD screen turned off. When we unplugged the white wire that connects the Open PCR Circuit Board to the 16 Tube PCR Block, the machine could not register or measure the temperature.
On October 18, 2012, our group first tested the Open PCR Machine. At first the machine seemed overwhelming in its design. However, after following the instructions and advice from peers and professors, we were able to determine how to properly setup, program, and run a simple test.
ProtocolsPolymerase Chain Reaction Polymerase Chain Reaction is a technology that amplifies a single piece of DNA. This technology works very similarly to the natural DNA replication cycle. One PCR cycle consists of three basic steps, denaturation, annealing and extension. In the denaturation step, heat (usually about 95 degrees Celsius) is used to separate the DNA into two strands. Then in the annealing step, the temperature is decreased to 50 degrees Celsius and the DNA primer, specific to the target sequence for that organism, anneal to the separated strand of DNA. The primers mark the beginning and the end of the targeted DNA sequence. Finally, the extension step required the temperature to be raised to 72 degrees Celsius so that the DNA polymerase is activated. The DNA polymerase begins synthesis at the DNA primer. This results in two double stranded target DNA sequences. The PCR cycle is repeated many times to amplify the targeted strand. There are typically many cycles that need to take place in the PCR in order to amplify a patient's DNA.
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Reagent | Volume | |||||
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Template DNA (20ng) | 0.2 μL | |||||
10 μM forward primer | 1.0 μL | |||||
10 μL reverse primer | 1.0 μL | |||||
GoTaq master mix | 50.0 μL | |||||
dH2O | 47.8 μL | |||||
Total Volume | 100.0 μL |
We ran a total of 8 samples. We made 3 samples from Patient 1 (ID#65685) was female and aged 58. We made 3 samples from Patient 2 (ID#58278) who was also female and aged 53. The remaining 2 samples were the positive and negative controls. The positive control for this experiment was the cancer DNA template and the negative control was the no DNA template.
Flourimeter Measurements
(Add your work from Week 3, Part 2 here)
Research and Development
Specific Cancer Marker Detection - The Underlying Technology
(Add a write-up of the information discussed in Week 3's class)
(BONUS points: Use a program like Powerpoint, Word, Illustrator, Microsoft Paint, etc. to illustrate how primers bind to the cancer DNA template, and how Taq polymerases amplify the DNA. Screen-captures from the OpenPCR tutorial might be useful. Be sure to credit the source if you borrow images.)
Results
(Your group will add the results of your Fluorimeter measurements from Week 4 here)
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