OUR TEAM
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|  Name: Mikayle Holm Role: Experimental Protocol Planner |  Name: Coleen Fox Role: Experimental Protocol Planner |
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
For PCR Protocol
| Supplied in the Kit
| Volume
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| PCR Reaction Mix (a.k.a. GoTaq Mix) |
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| Tag DNA Polymerase | enough for exp.
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| MgCl2, dNTP's, Forward and Reverse Primer | Ammounts are all suppliedd in kit
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| DNA Samples
| 50 μL each
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| Patients' DNA | DNA of Rabbit Eyeball
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| Negative control | Positive Control
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| SYBR GREEN1 | ' '
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| Supplied by User
| Amount
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| Lab Coat | 1 or 2
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| PCR machine made of steel with top that snaps | must clip shut!
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| Fluorimeter with built in camera and slides with dots farther apart | 12 new glass slides
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| micropipettes | at least 5 for mistakes
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| Eppendorf tubes | you will have a whole box, but will need one for each test
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PCR Protocol
BE CAREFUL NOT TO CROSS CONTAMINATE!!
1. Label Eppendorf Tubes with 1-17. Each tube should contain 50 microliters of one of the following substances:
| Test Tube
| Contents
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| 1 | Positive Control
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| 2 | Negative Control
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| 3 | Patient 1, Replicate 1
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| 4 | Patient 1, Replicate 2
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| 5 | Patient 1, Replicate 3
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| 6 | Patient 2, Replicate 1
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| 7 | Patient 2, Replicate 2
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| 8 | Patient 2, Replicate 3
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| 9 | Patient 3, Replicate 1
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| 10 | Patient 3, Replicate 2
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| 11 | Patient 3, Replicate 3
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| 12 | Patient 4, Replicate 1
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| 13 | Patient 4, Replicate 2
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| 14 | Patient 4, Replicate 3
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| 15 | Patient 5, Replicate 1
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| 16 | Patient 5, Replicate 2
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| 17 | Patient 5, Replicate 3
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2. Use properly labeled micropipette to place 17 samples into 17 properly labeled Eppendorf tubes already containing 50 microliters GoTaq mix. To see contents of GoTaq mix, see materials.
3. Place 17 Eppendorf tubes containing DNA and GoTaq Mix in Open PCR Machine. This PCR machine is made of steel in order to decrease safety hazards, and is able to hold more Eppendorf tubes. Therefore, all 17 Eppendorf tubes fit in this PCR. The top of the PCR machine snaps shut and can be clipped down to be less dangerous.
4. To make up for the more Eppendorf tubes in the PCR machine, each cycle is increased by 2 degrees Celsius and more power is needed to increase temperature, making the Thermal Cycler program:
Stage One: 1 cycle, 97 degrees Celsius for 180 seconds
Stage Two: 35 cycles, 97 degrees Celsius for 30 seconds, 59 degrees for 30 seconds, 74 degrees Celsius for 30 seconds
Stage Three: 74 degrees Celsius for 180 seconds
Final Hold: 6 degrees Celsius
5. When PCR is complete, proceed to DNA Measurement Protocol
DNA Measurement Protocol
BE CAREFUL NOT TO CROSS CONTAMINATE!!
1. With permanent marker, clearly number micropipettes. With permanent marker, number Eppendorf tubes at the top. There should be 17 labeled micropipettes and 17 Eppendorf tubes.
2. Transfer each sample from PCR Eppendorf tubes to labeled (1-17) Eppendorf tubes containing 400 microliters of buffer. Get all of the sample into the Eppendorf tubes.
3. Take specially labeled Eppendorf tube (18) with SYBR GREEN I and using specially labeled micropipette (18) place 2 drops on first two centered drops on fluorimeter slide. These dots are further apart on this fluorimeter slide in order to avoid contamination.
4. Take diluted sample from labeled Eppendorf tubes and place 2 drops on top of sample of SYBR GREEN I drop.
5. Turn light on, place beam so that it is passing through the drop.
6. Take picture with built in camera to avoid variability on smart phone camera settings.
7. Use micropipette properly labeled with black strip for waste to discard sample.
8. Repeat 3-7 for all samples (Eppendorf tubes 1-17), the DNA of rabbit eyeball (Eppendorf tube 19), and scintillation vial.
9. Generate pictures in ImageJ to analyze results.
Research and Development
Background on Disease Markers
Primer Design
Illustration
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BME 103 Fall 2012
