BME103:T130 Group 13: Difference between revisions
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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 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. The LCD screen displayed random numbers including -40 degrees Celsius (which is not possible because the Open PCR machine was not changing the temperature at that time). | 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. The LCD screen displayed seemingly random numbers including -40 degrees Celsius (which is not possible because the Open PCR machine was not changing the temperature at that time). | ||
Revision as of 16:52, 11 November 2012
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. The LCD screen displayed seemingly random numbers including -40 degrees Celsius (which is not possible because the Open PCR machine was not changing the temperature at that time).
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.
After the DNA has been through the thermal cycler, mix each new DNA sample with the the PCR master mix (Taq DNA polymerase, dNTP's, MgCl2, forward primer, and reverse primer) into 8 different Eppendorf tubes using separate pipettes to reduce contamination (see Table 1).
Flourimeter Measurements
After assembling the fluorimeter, you can now actually see if the target DNA was amplified in this experiment.
Research and DevelopmentSpecific Cancer Marker Detection - The Underlying Technology The reason that the cancer-associated sequence of r17879961 will produce a DNA signal while the non-cancer DNA sequence of the same SNP (single nucleotide polymorphism) will not produce a DNA signal lies in the arrangement of nucleotides at the molecular level. The lack of a DNA signal is due to the inability of the reverse primer to bind to the forward strand during the annealing phase of PCR. To detect the cancer-associated sequence of r17879961, the reverse primer AAC TCT TAC ACT CGA TAC AT is used. This is because the cancer-associated mutation is represented by a single nucleotide in a particular triplet: instead of the normal ATT, the middle T mutates into a C, thus rendering a triplet of ACT (which you can see in the reverse primer shown above). At the protein level, this mutation of 1 nucleotide changes the coded protein from isoleucine to threonine. As a result, the primer will not attach to the normal r17879961 DNA sequence as it will not have the corresponding base pairs (TGA) in the particular section of DNA that the mutated sequence would have. (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
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