BME103:W930 Group8: Difference between revisions
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'''Part 2: Flourimeter Protocol''' | '''Part 2: Flourimeter Protocol''' | ||
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'''Flourimeter Measurements'''<br> | '''Flourimeter Measurements'''<br> |
Revision as of 11:17, 7 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 | |||||||||||||||||||||||||||||||||||||||||
Group 8 PCR GurusLAB 1 WRITE-UP(Please finish by 11/7/2012) Initial Machine TestingThe Original Design Experimenting With the Connections When we unplugged part the LCD screen(part 3) from the arduino(part 6), the machine LCD display when black and ceased to work. When we unplugged the white wire that connects the Ardiono(part 6) to the PCR block (part 2), the machine read a temperature reading of -40˚C, indicating a malfunction.
(Write the date you first tested Open PCR and your experience(s) with the machine)
ProtocolsPart 1: PCR Protocol
Polymerase Chain Reaction (Polymerase Chain Reaction is used to amplify DNA. In order for this process to be successful, Template DNA must be replicated. A template of DNA consists of a strand in which the order of the nucleotides (bases) is known. With this information, it is possible to replicate the DNA strand by using heat, primers, and polymerase. The process begins by combining the DNA and the master mix. The master mix is composed of all of the necessary ingredients for the completion of the PCR process. The DNA is then placed into a PCR machine, or thermal cycler. The DNA is heated in the thermal cycler, allowing it to denature, or separate into two strands. The primers are then added to the template strand. They mark the start and end points of the specific sequance that is being targeted for replication. Next, the cycler is once again heated. This allows for the polymerase enzyme to activate and to begin adding nucleotides to the DNA strand. At the end of this process two strands are created (Essentially each strand of DNA has one side composed of new new nucleotides that are added by the polymerase and the other composed of the original nucleotides, thus allowing for a total product of two strands). The machine is then cooled and re-heated again. This cycle is generally repeated for 20-30 cycles, or until the desired amount of target DNA sequence strands is reached. The composition of the master mix is listed below. Steps to Run PCR
Part 2: Flourimeter Protocol Flourimeter Measurements (The flourimeter is used to___. The blue LED light is turned on to begin using the device. A camera phone is placed on to a designated tray. In order for the process to work, the flash settings on the camera phone must be turned off. Also the ISO must be 800 or higher and exposure must be set to its maximum. It helps to turn off auto focus as well. A slide is placed on to the flourimeter. On this slide a drop of water is inserted in the middle of the first two rows using a pipette. Two more drops are added to the first drop. The LED light is aligned with the drop and the flourimeter is then covered using a light box. The light box will remove excess light, allowing for images of the drop of water to be taken. The drop is then removed from the slide.The process of putting drops into the slide is then repeated using the next set of holes on the slide (Move the slide so that the new set of holes is in line with the blue light before putting the next drop onto the slide. For both parts of the process, each drop should be between 130 and 160 microliters.) The process of taking photos of the drop is then repeated using the same method previously presented. About three photos should be taken for each drop.
Note: The computer will recognize that it is a picture file and save it as Jpeg, unless otherwise specified. Research and DevelopmentSpecific Cancer Marker Detection - The Underlying Technology 3. Under Research and Development, explain why the rs17879961 cancer-associated sequence will produce a DNA signal and why a non-cancer DNA sequence will not. In other words, use the rs17879961 SNP to explain how the PCR detection method works (instead of the fictional sequences the professor used before). Our certain sequence rs17879961 is related to the susceptibility to Breast and Colorectal Cancer. In the case of PCR amplification and detection, the sequence for rs17879961 would be copied for by a primer. The primer starts the copying going forward and backward, thus the primers would be CCT for the 3' forward and GCC for the 3' backward. This primer would be used to detect the cancer sequence out of the DNA. The patient would have DNA extracted and prepped for PCR. This prep would include the insertion of the primer, dNTPs, taq Polymerase, solution and a salt, such as Mg2+. This solution would be inserted into the PCR machine to be heated/cooled/heated. During this fir heating process the DNA of the patient would be elongated and separated (along with the SNP for the cancer we are looking for).
(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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