SYBR Green Dye
SYBR green dye is a molecule that binds or stains a sample, in the case of this activity a double-stranded DNA or dsDNA. The SYBR green dye will give off a green colored-light when it is illuminated by a blue led light coming from the fluorimeter. This illumination reflects or signals that dsDNA is present in the sample and results to a positive signal; no green illumination means no dsDNA was present and therefore a negative signal.
Single-Drop Fluorimeter
The single-drop fluorimeter is a device that detects fluoresces. It includes a slide that has a smooth surface and a rough surface which is composed of craters that make it a superhydrophobic surface. Due to this superhydrophobic surfave, when the drop is placed on the rough side, the drop tends to have a beach ball or spherical shape which makes it easier to observe the signal. The device also includes a blue LED which excites the SYBR Green 1 in the drop.
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How the Fluorescence Technique Works
The fluorescence technique works by focusing a beam of blue LED light on the sample drop where the blue light, which has a short wavelength but high energy, increases the intensity and excites the SYBR Green 1 in the drop. When the blue light is focused on the drop, the drop absorbs the blue light and reflects or gives off a green colored light or signal.
Procedure
Smart Phone Camera Settings
Type of Smartphone: iPhone 5
Flash: No Flash
ISO setting: Auto
White Balance: Auto
Exposure: Auto
Saturation: Auto
Contrast: Auto
Type of Smartphone: Samsung Galaxy S3
Flash: No Flash
ISO setting: 800
White Balance: Auto
Exposure: 0
Saturation: Auto
Contrast: Auto
Calibration
Place the phone in the provided plastic cradle.
Adjust the position of the cradle with the phone so that the camera lines up with the center of the fluormeter where the illuminator is present.
If possible, focus the camera on the position of where the droplet would rest.
Measure the distance between the camera and where the droplet would be positioned.
Distance between the smart phone cradle and drop = 7.8cm
Solutions Used for Calibration
Calf Thymus DNA solution concentration (microg/mL)
Volume of the 2X DNA solution (uL)
Volume of the SYBR GREEN I Dye solution (uL)
Final DNA concentration in SYBR Green I 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
Adjust the micropipette using the volume adjustment knob so that it collects 80uL total in volume.
Attaching a new tip, collect 80uL of the SYBR green dye.
Release the SYBR green dye collected in between the two clear circles on the slide.
Dispose the tip and attach a new tip.
With the new tip, collect 80uL of a single concentration of the DNA sample and release the sample above the SYBR green dye so that the solutions mix.
Once the droplet is aligned with the illuminator of the fluorimeter, put the lightbox over with one flap up.
When the camera is refocused and the timer is fixed, start the camera and close the opened flap.
Repeat steps 2-7 two more times for one concentration.
Repeat the whole procedure for all concentrations. This will result in three tests of each concentration of DNA sample.
Data Analysis
Representative Images of Samples
Negative signal, 0 concentration of Calf Thymus DNA
Positive signal, 1 concentration of Calf Thymus DNA
BME 100 Fall 2013
