SYBR Green Dye
SYBR green dye is used in molecular biology as a nucleic acid stain. The dye generally binds to double-stranded DNA, while staining single-stranded DNA. The DNA-dye-complex that forms emits a green light (wavelength of approx. 520 nm) and absorbs blue light (wavelength of approx. 497 nm). SYBR green dye is commonly used in various methods of quantitative PCR.
Single-Drop Fluorimeter
A device that measures the fluoresence within a given drop of DNA solution. The quantity of fluorescence is generally directly related to the amount of florescent material in the given solution. The fluorimeter is capable of measuring the intensity and wavelength distribution of the spectrum that is emitted when the light passes through the DNA solution.
How the Fluorescence Technique Works
The fluorescence technique works by using a dye that makes the proteins glow a fluorescent green, showing whether the protein is either present or is not.
Procedure
Smart Phone Camera Settings
Model: HTC One
Flash: No
ISO setting: 100
White Balance: Fluorescent
Exposure: 0 (Default)
Saturation: 0 (Default)
Contrast: 0 (Default)
Calibration
We set the light and slides about 7 cm from the smartphone camera and we set the box over it to block out the light.
Distance between the smart phone cradle and drop = 7cm
Solutions Used for Calibration
Calf Thmus Dna solution concentration (microg/mL)
Volume of the 2X DNA solution (microliters)
Volume of the SYBR GREEN I Dye solution (microliters)
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
0.125
80
80
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Placing Samples onto the Fluorimeter
Place the slide in its designated place, smooth side down, into the single droplet Fluorometer device.
Micro-pipette 80µL of the SYBR green dye and place it in the second row of the middle column of the slide.
Next carefully Micro-pipette 80µL of desired concentration of calf thymus DNA on top of the SYBER green dye onto the slide.
The total amount of SYBR green dye/ calf thymus DNA mixture will be 160µL
Start test by taking a photo of the droplet.
Data Analysis
Representative Images of Samples No DNA
With DNA
Image J Values for All Samples
Calf Thymus DNA Concentration (FINAL), μg/mL
Image Number
AREA
Mean Pixel Value
RAWINTDEN OF THE DROP
RAWINTDEN OF THE BACKGROUND
2.5
1
33691
132.209
4454265
4454265
2.5
2
33840
131.203
4439919
4439919
1
1
36244
79.188
2870091
2870091
1
2
43552
79.813
3476030
3476030
0.5
1
37294
37.722
1406812
1406812
0.5
2
31940
38.819
1239877
1239877
0.25
1
43038
23.1
994189
994189
0.25
2
35870
27.141
973557
973557
0.125
1
41800
6.658
278307
278307
0.125
2
39159
5.107
199977
199977
0
1
44228
6.221
275159
275159
0
2
39651
7.252
287535
287535
Note: Only two images needed per Dr. Garcia since there were initial problems with the HD camera
BME 100 Fall 2013
