SYBR Green Dye
SYBR Green Dye is a molecular dye that is not fluorescent in water or small amounts of single strand DNA, but is very fluorescent with dsDNA (double-stranded DNA).
Single-Drop Fluorimeter
A Single-Drop Fluorometer is used to detect and measure fluorescence using the amount of fluorescent material, intensity, and wavelength distribution of emission spectrum.
The Fluorimeter
The Fluorimeter pictured above was the device used for this lab. The gap in the middle-right is for a hydrophobic slide to be inserted and then slid into position. The desired position results in the beam of light being passed through the drop of liquid. The toggle switch on the left controls the beam of light.
How the Fluorescence Technique Works
The fluorescence technique works by shining a light of a shorter wavelength to be absorbed by a molecule. This will excite the molecule and it will emit a brighter light with a higher wavelength.
Procedure
Smart Phone Camera Settings
Type of Smartphone: Samsung Galaxy S1
Flash: OFF
ISO setting: 150 Auto
White Balance: "Auto"
Exposure: "Auto"
Saturation: 0
Contrast: 0
This was the only smartphone used throughout the experiment.
Calibration
First, we placed the smart phone in the cradle given to us, then adjusted the vertical distance by adding slides beneath the fluorimeter. After getting the height between the fluorimeter and the camera in line, we placed the phone to focus on the drop and in return capture a clear image.
'
Distance between the smart phone cradle and drop = 4.5 centimeters.
Solutions Used for Calibration
Calf Thymus DNA Solution Concentration (micro/mL)
Volume of the 2X DNA Solution (microliter)
Volume of the SYBR Green I Dye Solution (microliter)
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
blank
Placing Samples onto the Fluorimeter
Using a micro-pipetter, place 80 microliters of the SYBR Green I dye in the middle of the first row of the slide.
Add 80 microliters of the desired concentration of calf-thymus solution or water to the 80 microliters of SYBR Green I dye.
Make sure the solution of 160 microliters is aligned with the blue LED light!
Set up the timer function on the smartphone being used, and place it into the cradle (used to avoid moving, shaking, or bumping the phone)
Place the fluorometer and camera in the cradle into the light box. The light box is used to keep as much light as possible from affecting the data.
Record the distance between the fluorometer and lens of the camera on the smartphone in case the apparatus must be moved before the experiment is over.
Making sure the camera is focused, take 2-3 pictures of the drop on the fluorometer.
Remove the light box.
Use the micro-pipetter to remove the 160 microliter drop from the surface of the slide.
Repeat these steps two more times for the same concentration, using different areas on the same slide.
Go through these steps three times for every desired concentration, using different slides for each concentration.
Data Analysis
Representative Images of Samples The picture above represents the negative control, which is SYBR Green without DNA. The picture above represents the positive control, which is the SYBR Green past the point of saturation.
Image J Values for All Samples
'
Area
Mean
Min
Max
IntDen
RawIntDen
'
IntDen
Concentration ug/mL
1
42076
102.625
1
255
4318055
4318055
4318055
2.50
2
42948
100.491
1
255
4315896
4315896
4315896
2.50
3
42948
105.926
4
255
4549299
4549299
4549299
2.50
4
42948
91.052
3
219
3910510
3910510
3910510
1.00
5
42948
85.141
0
255
3656651
3656651
3656651
1.00
6
42948
89.729
4
255
3853672
3853672
3853672
1.00
7
42948
60.908
0
255
2615887
2615887
2615887
0.50
8
42948
60.783
0
255
2610502
2610502
2610502
0.50
9
42948
63.747
19
255
2737802
2737802
2737802
0.50
10
42948
33.408
0
255
1434791
1434791
1434791
0.25
11
42948
33.408
0
255
1434791
1434791
1434791
0.25
12
42948
39.597
0
255
1700604
1700604
1700604
0.25
13
42948
30.104
0
249
1292917
1292917
1292917
0.125
14
42948
38.351
3
233
1647118
1647118
1647118
0.125
15
42948
34.052
0
238
1462475
1462475
1462475
0.125
16
42948
18.085
0
255
776731
776731
776731
0.00
17
42948
13.576
0
230
583066
583066
583066
0.00
18
42948
16.245
0
234
697697
697697
697697
0.00
Fitting a Straight Line
The graph above establishes the relationship between the integrated density (INTDENS) readings before background correction and the calf thymus DNA concentration in micrograms per milliliter. The integrated density is the sum of independent, noninteracting subsystems that hold proportional to the amount of material in the substance paralleled in observation of the direct relationship between the integrated density and the concentration of the calf thymus DNA.