BME103:T930 Group 4

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 TEAM Name: Alex Hoang Role(s): Open PCR Machine Engineer; DNA Measurement Operator Name: Bobby Ryan Role(s): Open PCR Machine Engineer; ImageJ Software Processor Name: Sabrina Freeman Role(s): Experimental Protocol Planner; Sample Preparation & Application Name: Sheanah Conneen Role(s): Experimental Protocol Planner; Data Compiler & Analyzer Name: Hayden Hilliard Role(s): Research and Development Scientist LAB 1 WRITE-UP Initial Machine Testing The Original Design https://myasucourses.asu.edu/courses/1/2012Fall-T-BME103-86053-86055-86054/groups/_185910_1//_7694078_1/Open%20PCR.png Experimenting With the Connections When we unplugged PCB Board of LCD from PCB of open PCR Circuit Board, the LCD display would turn off. When we unplugged the white wire that connects PCB of open PCR Cicuit Board to the temperature sensor wire, the temperature reading of the machine dropped drastically. Test Run 10/25/12 Testing the Open PCR for the first time was more complicated than we expected. Our Open PCR had technical difficulties that hindered its performance. Our samples took over 2 hours to cycle completely compared to other Open PCRs which took about 1 1/2 hours. In the end, the machine performed its task successfully. The software interface of the Open PCR was easy to understand and use. The LCD screen display also made it easy to monitor the progress of the test. Protocols Polymerase Chain Reaction Polymerase Chain Reaction (PCR) is a process used to amplify DNA by making millions of copies of a particular sequence. Prepare DNA samples and reagents in their respective containers. Create three copies of each original DNA sample. Be sure to label each container explicitly. Place samples in the PCR machine. Start the PCR machine. The samples will be heated to 95°C to unwind the helix. The temperature will drop to 57°C to activate the primer and strand pairing. The temperature will then rise to 72°C to activate the DNA polymerase for replication. Remove the sample from the PCR machine and store them until it is time to take flourimeter measurements. The PCR master mix is GoTaq® Colorless Master Mix, a premixed ready-to-use solution containing bacterially derived Taq DNA polymerase, dNTPs, MgCl2 and reaction buffers at optimal concentrations for efficient amplification of DNA templates by PCR. Results Volume Template DNA (20 ng) 0.2 μL 10 μM forward primer 1.0 μL 10 μM reverse primer 1.0 μL GoTaq master mix 50.0 μL dH2O 47.8 μL Total Volume 100.0 μL Eight samples were ran. The first sample was a positive control for the gene being replicated. There were three samples from patient number 87998, a 48 year old female patient. There was also a negative control for the gene being replicated. There were three samples from patient number 21822, a 45 year old male patient. Flourimeter Protocol In order to prep for our measurements we took the following steps: We numbered our pipettes and our Eppendrof tubes so that we would only use it for one specific sample. We then transferred each sample into and Eppendorf tube containing 400 ml of buffer and labeled it with the number of the sample. Next we took the Eppendrof tube containing SYBR Green I and placed two drops on the first two centered drops. Then we took the diluted sample and placed two drops on top of the SYBR GREEN I solution drops. We aligned the light so that it was going through the drop. After setting the iPhone to the proper settings, we took pictures of each sample using the light box. In order to analyze our images, we loaded them on IMAGEJ. After making sure the settings were correct, we drew ovals around the green drop images and hit ANALYZE>MEASURE We recorded this data and continued on to get the background readings by drawing ovals of the same size as before in the green file to get the "noise". We then recorded these results. 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 Sample Integrated Density DNA μg/mL Conclusion PCR: Negative Control 639,218 1.654 negative PCR: Positive Control 405,796 1.107 positive PCR: Patient 1 ID 87998, rep 1 332,666 0.8606 positive PCR: Patient 1 ID 87998, rep 2 716,695 1.854 positive PCR: Patient 1 ID 87998, rep 3 868,481 2.247 positive PCR: Patient 2 ID 21822, rep 1 272,706 0.705 negative PCR: Patient 2 ID 21822, rep 2 99,377 0.257 negative PCR: Patient 2 ID 21822, rep 3 205,460 0.532 negative KEY Sample = Integrated Density = DNA μg/mL = Conclusion =