OUR TEAM
 Name: Benjamin Hook Protocol |
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LAB 2 WRITE-UP
Thermal Cycler Engineering
Our re-design is based upon the Open PCR system originally designed by Josh Perfetto and Tito Jankowski.
System Design
Key Features
Instructions
Protocols
Materials
| Supplied in Kit
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| Open PCR Machine
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| (32) Plastic Test Tubes to Fit into PCR Machine
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| PCR Power Adapter
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| USB Cable
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| Flourimeter
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| (8) Glass Slides
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| Supplied by User
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| Low Retention Adjustable Pipette with Disposable Pipette Tips
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| DNA Solution
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| Positive Control DNA Soltution
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| Negative Control DNA Solution
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| What the DNA Solution Should Consist Of
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| 1 Micro-Liter Forward Primer
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| 1 Micro-Liter Reverse Primer
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| 50 Micro-Liter GoTaq Master Mix
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| .2 Micro-Liter Patient's DNA (Or Controls)
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| 47.8 Micro-Liter Distilled Water
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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 good results).
Step 4: Using the Pipette, transfer 32 samples of the patients DNA into each test tube. You should only use 1 Pipette tip for this part of the process.
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.
Step 2: Label the pipettes to match up with the correlating test tubes.
Step 3: Use one pipette for the SYBR Green and one pipette for waste.
Step 4:
Research and Development
Background on Disease Markers
Primer Design
Illustration
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BME 103 Fall 2012
