BME103:T930 Group 2

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 AWESOME TEAM Ryan Sullivan Research Development Scientist Miriam Acosta PCR Machine Engineer Ryan Keeney PCR Machine Engineer Juliana Ramos Experimental Protocol Planner Aaron Cornejo Experimental Protocol Planner LAB 1 WRITE-UP Initial Machine Testing Open PCR. The Original Design An Open PCR machine is a thermo-cycler used to cycle DNA. By rapidly and precisely changing the temperature through a heating plate and cooling fan, the machine manages to oscillate the temperatures of the DNA samples. The low-cost machine runs through software that is user-friendly and initiates the process once the heated lid is properly closed on top of the DNA samples. The samples are placed in the designated sample holders and the computer program commences the process. The PCR goes through a cycle of heat changes for a specified duration of time in order to facilitate the replication and amplification of DNA sample. Experimenting With the Connections Unplugging the LED interface cable from the processing chip caused the display of the machine to turn off. The blank screen meant that the machine stopped sending data to display. Unplugging anything from the circuit board causes it not to work because no electricity can be sent. When the white wire was disconnected from the circuit board, the PCR machine discontinued to record temperatures correctly. Thus making the Open PCR machine impractical for its actual function. Test Run (Write the date you first tested Open PCR and your experience(s) with the machine) Protocols Polymerase Chain Reaction Reagent and Volumes using PCR. Patient information. Flourimeter Measurements Droplet of water on hydrophobic slide. Proper set up. Flourimeter Assembly Instructions: 1. Gather materials (PCR Machine, tools optional) 2. Remove the lid from black box and flip-flop the box 3. Open the front flap of the black box. 4. Place hydrophobic/glass slide 5. Using pipette to obtain droplets of SYBR green dye 6. Proceed to squeeze two drops of dye on the slide. 7. Obtain DNA sample. 8. Add DNA sample to the SYBR green dye. 9. Set up glass and box as shown in the picture. 10. Make sure the blue LED light shines through the droplet. 11. Take picture with smartphone (should be set up as indicated in step 9) 12. Double check thatthe stand and the flourimeter is covered by the box to make sure not light comes in. 13. Take a well focused photo with smartphone. 14. Finally, use ImageJ to obtain information from the picture. Research and Development Specific Cancer Marker Detection - The Underlying Technology The r17879961 DNA was sequenced to determine the chain of nucleotides that it is composed of. A section of this DNA shows a change in the allele sequence; one which might indicate cancer. In the sequence, a change from "ATT" to "ACT" is the specific indication marker for cancer. The artificial primer that is introduced to the DNA chain is meant to be a complimentary strand that matches up with the "ACT" and surrounding nucleotide sequence ("TGA"). When the primer attaches successfully, it allows the Taq polymerase to finish attaching nucleotides to the sequence. The number of strands will be doubled in this case, and by using the PCR replication machine, the sample will be heated and cooled in specific intervals. These intervals are meant to manipulate the DNA and do things such as separate a strand, cool it down so it is able to bind, as well as stretch it out. This process will, ultimately, result in exponential DNA replication. If the primer does not attach successfully, then this means the person does not have a cancer-indicating sequence, and it will, therefore, not replicate the DNA exponentially. If the DNA replicates exponentially, then the fluorescent dye will show, and this would indicate a cancerous genome. After the PCR machine replication, the samples were put on a slide and combined with Sybr Green indicator. They were then placed in a dark box and a picture was taken of each sample. The samples pictures were uploaded onto the computer and we were able to use software to measured the amount of green fluorescent dye that was in the replicated DNA. The data in the section below describes the data and the parameters that indicate cancer using this information. The illustration below shows the PCR process when the primer attaches to the DNA and the Taq polymerase and is able to complete the DNA chain, in coordination with the PCR machine temperature cycles. It is shown that throughout the process, the DNA replicates at an exponential rate. Results Sample Integrated Density DNA μg/mL Conclusion PCR: Negative Control E6 F6 G6 PCR: Positive Control E7 F7 G7 PCR: Patient 1 ID #####, rep 1 E8 F8 G8 PCR: Patient 1 ID #####, rep 2 E9 F9 G9 PCR: Patient 1 ID #####, rep 3 E10 F10 G10 PCR: Patient 2 ID #####, rep 1 E11 F11 G11 PCR: Patient 2 ID #####, rep 2 E12 F12 G12 PCR: Patient 2 ID #####, rep 3 E13 F13 G13 KEY Sample = Integrated Density = DNA μg/mL = Conclusion =