BME103:T930 Group 11 l2

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: Benjamin Hook Protocol Name: Jacqueline Janssen Protocol Name: Samantha Boccasini Role(s) Name: Tony Nguyen R&D Name: Ryelle Pattuinan R&D Name: Team member Roles(s) LAB 2 WRITE-UP Thermal Cycler Engineering System Design Our re-design is based upon the Open PCR system originally designed by Josh Perfetto and Tito Jankowski. Our new design incorporates some new designs such as software, screen zize, number of testing tube lots, as well as size of heating lid. All of these alterations are made to make the Open PCR system more efficient in terms of its operating system and user-friendly features. Key Features Our most major change to the Open PCR System is the change we made to the read-out screen on the top of the device near the heating lid. This change actually affects a few major components of our system. Not only did we move the screen to the side of the machine, rather than the top, but we also optimized the size of it. This size-change allows users to see the read-outs clearer. We also eliminated the need for a computer (or any outside device, that is) as this new larger screen will also be able to control the machine. Now the user is able to input cycles, temperature, etc. right on the screen instead of needing to plug it into a separate system. This allows for better portability and easier use. We also changed the space of the testing tubes so now more tubes can be tested at once. To do this we lengthened the plate as well as the heating lid entirely across the top of the machine. Removing the screen from this part of the Open PCR System also allowed for this change. Instructions Protocols Materials Supplied in Kit Open PCR Machine (32) Plastic Test Tubes to Fit into PCR Machine PCR Power Adapter USB Cable Flourimeter (8) Glass Slides SYBR Green (200 mL) Measurement Buffer (800 mL) Black Box Positive Sample (50 mL) Eppendorf Tubes (40) Supplied by User Low Retention Adjustable Pipette with Disposable Pipette Tips DNA Solution Positive Control DNA Soltution Negative Control DNA Solution 1 Smartphone Pair of Gloves 1 Lab Coat Water Sample What the DNA Solution Should Consist Of 1 Micro-Liter Forward Primer 1 Micro-Liter Reverse Primer 50 Micro-Liter GoTaq Master Mix .2 Micro-Liter Patient's DNA (Or Controls) 47.8 Micro-Liter Distilled Water PCR Protocol Step 1: Download the Open PCR Software onto Computer Step 2: Plug in and turn on the Open PCR machine, connect the USB cable to your computer Step 3: On the machine interface, select "DNA replication" and then choose desired number of cycles (at least 30 for quality results). Step 4: Using the Pipette, transfer 30 samples of the patients DNA into each test tube. You should only use 1 Pipette tip for this part of the process. Also transfer the positive and negative control into separate test tubes. Step 5: Next, transfer the forward and reverse primers into each of the 32 test tubes. 2 Pipette tips should be used in this part of the process: 1 for all of the forward primer transfers, and 1 for all of the reverse primer transfers. Step 6: Using a new pipette tip, transfer the GoTaq Master mix into each of the 32 test tubes. Step 7: Dilute the 32 solutions by filling the remainder of the test tube with distilled water. Step 8: Carefully Label Each test tube with a sharpie making sure that the positive and negative controls are clearly marked. Step 9: Open the heated lid of the Open PCR machine and place the test tubes into the designated slots. Close the lid. Step 10: Make sure everything is properly connected and then choose "begin replication" on the interface. Step 11: Check to make sure that the computer is correctly receiving the information, if not: stop the cycle, check the USB cord, and try again. DNA Measurement Protocol Step 1: When replicating is finished, remove the 32 tubes from the PCR Machine, as well as the positive and negative controls. Step 2: Transfer DNA samples to eppendorf tube containing 800 mL of buffer. Use a new tip on the pipette for each transfer of DNA to avoid contamination. Do the same for all the controls. Step 3: Using the rough side of the slides, place two drops over two holes on the slide of SYBR green. Carefully, using a new tip of a pipette, transfer two drops of a DNA sample on the SYBR green. Step 4: Place the slide in the black box directly in the ray of the light. Step 5: Take a photo of the slide using the smart phone. Step 6: Using a new pipette tip that is marked for waste, remove the sample from the slide and dispose of it. Step 7: Repeat steps 3-6 using a new pipette tip for each new sample or control. Step 8: Analyze the results, knowing that the samples that glow green are the positive ones. Research and Development Background on Disease Markers Primer Design Illustration