SBYR Green I dye is a cyanine dye used as a nucleic acid stain. When bound to DNA it absorbs blue light and emits green light. The stain binds to double stranded DNA (dsDNA) at very high levels and binds to single stranded DNA at much lower levels. This allows us to measure the amount of double stranded DNA while getting minimal noise on our signal from the presence of single stranded DNA. It can also stain RNA at lower levels, but this is not important for our experiment (in which we know our sample does not contain RNA). It is also the most sensitive stain available for detecting double stranded DNA during PCR.
Single-Drop Fluorimeter
This device is used to excite the stained DNA molecules in order to generate the designed signal, a green light, for us to capture with our camera. The single-drop fluorimeter is designed to hold a single drop of the sample and pass the wavelength ~497nm of light required to excite the stained DNA molecules within the sample.
How the Fluorescence Technique Works
A droplet is placed on the hydrophobic slide which allows the droplet to hold its spherical shape. The single droplet is then exposed to a blue light beam to excite the stained molecules which proceed to emit green light (our signal). We capture this signal (green light) with our smartphone camera. In theory, the amount of signal captured by our camera should be proportional to the concentration of DNA in the sample (the pictures must be filtered so that only the amount of green light captured is taken into consideration). Therefore, once we have calibrated our camera with known concentrations of DNA we should be able to compare signal strengths (green light emittance) from unknown DNA concentration samples with our calibration data to accurately estimate the DNA concentration in that sample.
Procedure
Smart Phone Camera Settings
Type of Smartphone: Samsung Galaxy SIII
Flash: OFF
ISO setting: 800
White Balance: Auto
Exposure: +2 (maximum)
Saturation: Auto (no manual options)
Contrast: Auto (no manual options)
Calibration
The camera was placed upright in the supplied stand. The camera lens was positioned 6cm from the droplet in order to get the closest view possible while still being within the focus range of the camera. The height of the fluorimeter was adjusted so that the lens of the camera was at the same height as the droplet to ensure a full side view.
Distance between the smart phone camera lens and drop = 6cm
Solutions Used for Calibration
Calf Thymus DNA Solution Concentration (µg/mL)
Volume of the 2X DNA Solution (µL)
Volume of the SYBR Green I Dye Solution (µL)
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
Place a clean slide with the hydrophobic side up into the slot in the Fluorimeter.
Turn on the Fluorimeter.
Use a micro-pipette to transfer 80 µL of SYBR Green I Dye and 80 µL of sample (either water or DNA solution) onto the slide to form a single droplet.
Move the slide until the droplet is directly in line with the blue light beam (if not so already).
Position camera at the predetermined position (see "Calibration") and set a countdown timer on the camera.
Focus the camera on the droplet and start the countdown timer.
Cover the entire apparatus with the supplied box to minimize external light and wait for the camera to capture the picture.
Remove the box, open the recently captured picture and rename it to something that represents the sample DNA concentration and picture number.
Remove the 160 µL droplet from the Fluorimeter slide with a micro-pipette and remove any remaining liquid with a paper towel.
Repeat the above steps two more times to get a total of three pictures of three different droplets of the same DNA concentration.
Repeat the above steps for each sample.
Data Analysis
Representative Images of Samples
The picture below shows the control case where no DNA is present in the sample
The picture below shows a test case where DNA is present in the sample
Image J Values for All Samples
CT DNA Final Concentration (µg/ml)
Area
Mean Pixel Value
RAWINTDEN Drop
RAWINTDEN Background
Corrected INTDEN
0
63850
9.801
625816
59414
566402
0
66618
14.951
996018
79261
916757
0
63749
8.569
546237
63420
482817
0.125
69214
8.492
587772
80722
507050
0.125
67620
21.642
1463410
93467
1369943
0.125
50109
8.395
420689
7013
413676
0.25
63624
30.175
1919850
65755
1854095
0.25
54968
31.24
1717180
58722
1658458
0.25
62254
34.8
2166468
13405
2153063
0.5
64057
76.566
4904612
69973
4834639
0.5
64966
55.922
3633024
15391
3617633
0.5
69714
54.787
3819397
13548
3805849
1
72788
89.233
6495124
62681
6432443
1
71872
89.702
6447038
72261
6374777
1
68364
87.689
5994752
14063
5980689
2.5
74024
124.657
9227622
16445
9211177
2.5
75904
130.099
9875048
84898
9790150
2.5
83280
127.561
10623306
76871
10546435
Fitting a Straight Line
The below graphs illustrate the linear correlation between dsDNA concentration and fluorescence. The first graph shows the raw fluorescence data plotted against the known dsDNA concentrations, and the second graph shows the corrected fluorescence data (raw integrated density minus background integrated density) plotted against the known dsDNA concentrations.