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Our re-design is based upon the Open PCR system originally designed by Josh Perfetto and Tito Jankowski.
System Design
Key Features
Instructions
Protocols
Materialsl
PCR Protocol
1.) The DNA samples were heated to ninety-five degrees Celsius (95°C) for one (1) minute to unzip the two single strands.
2.) They were then cooled to fifty-seven degrees Celsius (57°C) for ten (10) seconds and the primers were attached to their matching sequences.
3.) They were then heated back to seventy-two degrees Celsius (72°C) for ten (10) seconds and polymerase extended the DNA strands by attaching the correct free nucleotides in order on the single strands.
GoTaq Mix Components For 100μl reaction volume:
2X GoTaq Colorless Master Mix
10 μM upstream primer
10 μM downstream primer
DNA template
Nuclease-Free Water to
Reagent
Volume
Template DNA (20 ng)
0.1μL
10μM forward primer
0.5μL
10μM reverse primer
0.5μL
GoTaq master mix
25.0μL
dH2O
23.9μL
Total Volume
50.0μL
DNA Measurement Protocol
Fluorimeter Setup
1.) The lid was first taken off the the box and one of its sides was unbuttoned in order to create a flap.
2.) The box was the flipped upside down in order to create a dark environment for the camera.
3.) A hydrophobic slide was then inserted into the flourimeter.
4.) Finally, the camera phone was placed in the stand.
Fluorimeter Measurements
1.) Label transfer pipettes and tubes
2.) Transfer each sample separately into tube containing 400μl of buffer
3.) Take the specifically labeled tube containing SYBR GREEN 1 and place 2 drops on the first 2 centered drops
4.) Place 2 drops of diluted sample on top of the SYBR GREEN 1 drop
5.) Align light through drop
6.) Take pictures using light box
7.) Repeat for each sample.
8.) Run water as BLANK using same procedure
ImageJ Instructions
1.) Open ImageJ
2.) Click ANALYZE tool bar and select SET MEASUREMENTS
3.) Select AREA, MEAN GREY VALUE, and INTEGRATED DENSITY
4.) Upload image to ImageJ
5.) Select IMAGE then COLOR and then SPLIT CHANNELS
6.) Only use green channel
7.) Use OVAL tool and select the entire drop of liquid
8.) Go to ANALYZE and then MEASURE
9.) Drag circle to the background of the image
10.) Record results
11.) Repeat if necessary