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
The following materials are provided in the PCR and Fluorimeter kit:
Supplied in Kit
Amount
Open PCR Machine
1
Fluorimeter
1
Phone Stand
1
Box
1
Superhydrophobic Slides
10
CD Software
1
The following materials will need to be supplied by the user:
Supplied by User
Amount
Eppendorf tubes
10
Pipettes
12
Pipette Tips
8
Power source
1
Smartphone with Camera
1
ImageJ Software*
1
PCR Master Mix
800 μL
SYBR Green Solution
10 mL
DNA Samples
6
DNA Positive Control
1
DNA Negative Control
1
Calf Thymus DNA Sample
1
Water
100 mL
Plastic Tube-holding Grid
1
Note that the PCR machine does not require a computer but ImageJ does. A further improvement to this technology would be to design an app for ImageJ that can be used on smartphones.
PCR Protocol
In order to perform PCR, the samples must first be prepared. This is done by adhering to the following steps:
1. Open up all Eppendorf tubes that will be needed to perform the PCR. Place them in the plastic grid to hold them.
2. Fill each Eppendorf tube with 50 μL of the PCR Master Mix using a pipette.
3. Using a distinct, clean pipette tip per sample (including positive and negative controls), fill each tube with one DNA sample. Be sure to close each tube as you fill it with the DNA sample and label the lid. Throw away each pipette tip and place a new one on each time you are going to use a different sample to avoid contamination.
Now that the samples have been prepared, the PCR machine must then be configured to the specif cycling and temperature needs of your experiment.
1. Turn on the Open PCR machine and press the forward arrow key to select PCR.
2. Set the lid temperature to 100°C and press the forward arrow key when done.
3. Set the initial temperature to 95°C. Press the forward arrow key and select 3 for the number of minutes to hold the tubes there.
4. Set the number of cycles to 30 and press the forward arrow key.
5. For the first temperature, set it to 95°C, press the forward arrow key, and set it for 30 seconds.
6. Select the second temperature of the cycle to be 57°C, press the forward arrow key and set it to 30 seconds.
7. Select the third temperature of the cycle to be 72°C, press the forward arrow key and set it to 30 seconds.
8. Set the final temperature to 72°C, press the forward arrow key, and select 3 minutes.
9. Set the hold for the tubes to be 4°C.
10. When ready, place the test tubes into the machine, close the lid and twist the top to hold it down, and select the forward arrow key to start the PCR. Note that if a mistake was made you may use the backward arrow key to scroll through the different settings you selected and alter as necessary.
BME 103 Fall 2012
