BME103:T130 Group 10

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: Jeffery Ramirez Role: Protocol planner Name: Tyler Tamasauckas Role:R&D Specialist Name: Alexander Baldwin Open PCR Machine Engineer Name: student Role(s) Name: student Role(s) Name: student Role(s) LAB 1 WRITE-UP Initial Machine Testing The Original Design (Add image of the full OpenPCR machine here, from the Week 3 exercise. Write a paragraph description for visitors who have no idea what this is) The OpenPCR machine is designed to isolate and replicate certain sequences of DNA through heating and cooling the samples. There are several parts to it, most of which can only be seen if one of the outside walls is taken out. The samples of DNA are placed in small tubes and are then placed in the heating area, where they are repeatedly heated and cooled during several cycles to achieve the desired result. The time for a cycle is usually a few minutes, and there are usually several cycles in a run, so it may last over an hour. Experimenting With the Connections When the circuit board (part 6) was unplugged from the display (part 3), the display turned off and no longer registered any signal. The display was no longer functional, but turned on again once the wire that previously connected the two was plugged back in. When the circuit board was unplugged from the heating element, the heating element was no longer able to control its temperature. Although it still had power, it was not being controlled by the circuit board. Test Run The first test run completed on the OpenPCR machine was on October 25, 2012. The test was successful, and was completed in roughly the time expected, give or take a few minutes. Once the settings were put to the correct levels, the set up was quite simple. The samples were placed inside the heating unit, then the test run started. It took over an hour, but was completed successfully with no malfunctioning pieces of equipment. Protocols Polymerase Chain Reaction 1.)HOW PCR WORKS The Polymerase Chain Reaction machine, PCR for short, works by cycling DNA at different temperatures to amplify it, so it can be compared with other DNA. This is done by first denaturing the DNA, which happens by the PCR heating up, which causes the hydrogen bonds to break, resulting in the DNA strands breaking apart. The PCR then cools down, which allows a primer to bind to the target DNA. In the third step, the machine is heated back up so an enzyme can rebuild the DNA. In the final step, a fluorescent dye binds to the new double stranded DNA. This process is repeated many times, until there is a big enough DNA strand to be analyzed. 2.)STEPS TO AMPLIFY DNA The actual steps for PCR are quite simple. Step 1.) The PCR lid is heated to 100°C and the tubes are heated to 95°C. This step is ran for 3 minutes. Step 2.) The PCR is then set to run 30 cycles. Each cycle consists of heating to 95°C for 30 seconds, cooling to 57°C for 30 seconds, and finally, heating to 72°C for 30 seconds. Step 3.) The PCR is then held at 72°C for 3 minutes. Step 4.) The PCR is then kept constant at 4°C 3.)COMPONENTS OF PCR MIX 4.)TABLE OF REAGENTS AND VOLUMES USED 5.)SAMPLE DESCRIPTIONS Fluorimeter Measurements 1.)PHOTO OF SETUP 2.)STEPS TO ASSEMBLE THE FLUORIMETER To assemble the Fluorimeter, there are a few easy steps that need to be followed. The first step is to unbutton the front flaps and open the lid. Take out the interior contents which include the slide that the liquid is put on and the cell phone stand. Once this is done, close the lid so that only the front is open. Place your liquid on the slide, turn on the light and place it inside the box. Then put a cell phone with a camera in the cell phone stand. Align the camera with the drop of liquid on the slide. After this step, the Fluorimeter is set up. Refer to the image above for a photo representation of the set up. 3.)USING IMAGE J 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 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 =