SYBR Green Dye
SYBR Green Dye is a type of dye that is used to highlight when dsDNA is present because that is when it fluoresces the strongest. The dye fluoresces weak with water or single strands of DNA.
Single-Drop Fluorimeter
The Single-Drop Fluorimeter (as pictured above) is a custom device designed for the purpose of finding when a double-stranded piece of DNA is present. It fluoresces double-stranded DNA fragments in a green color, and does nothing to a solution without the double-stranded piece of DNA.
How the Fluorescence Technique Works
The process had several different variables that were chosen for very specific reasons. The slide to be used was chosen because it had a rough surface with glass circles exposed in it, causing the solution to concentrate at those locations and be easier to examine. The DNA dye chosen for the experiment was used because it was not completely soluble, which allowed for the light to glow instead of causing a reflective light off of the drop.
Procedure
Smart Phone Camera Settings
Type of smart phone used and adjusted camera settings.
The phone was set up in the phone stand so that the camera was focusing on the drop. It was zoomed in far enough to notice the color of the drop and how it changed with each concentraton change.
Distance between the smart phone cradle and drop = 3.7 cm
Solutions Used for Calibration[See worksheet page 5]Image J Values for All Samples[See worksheet page 5]
Calf Thymus
DNA solution
concentration
(microg/mL)
Volume of the
2X DNA
solution
(µL)
Volume of the
SYBR GREEN I
Dye solution
(µL)
Final DNA
concentration in
PicoGreen Assay
(ng/mL)
5
80
80
2.5
2
80
80
1
1
80
80
0.5
0.5
80
80
0.25
0.25
80
80
0.125
0
80
80
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Placing Samples onto the Fluorimeter Materials
SYBR green
Pipette
Water
Hydrophobic Tray
Smartphone
Cradle
Box (For Cover)
Plastic Base
Step 1: Calibrate your smartphone to ensure the proper imaging environment.
Step 2: Place the pre-determined concentration droplet of SYBR green onto the hydrophobic tray between the initial 2 ruts.
Step 3: Make sure the droplet is centered on the green light to ensure proper imaging.
Step 4: Place the hydrophobic tray onto the cradle.
Step 5: Set up the smartphone on the plastic base a fixed distance away from the hydrophobic tray.
Step 6: Level the view of the camera to show the droplet in a horizontal perspective.
Step 7: Place the box over the contraption to create a dark room.
Step 8: Take the picture of the droplet in the closed environment.
Step 9: Repeat the initial steps for all the sample trials.
Step 10: Use the pictures to analyze the affect of the concentration of SYBR green in the droplet samples.