SYBR Green Dye
SYBR Green I dye is a cyanine dye that binds to DNA and is commonly used as a nucleic acid stain. The dye absorbs blue light (497 nm) and emits green light (520 nm). While it preferentially binds to double stranded DNA, the dye will also bind to single strand DNA with the reslt being somewhat lower performance. Finally, SYBR Green I dye is commonly used in PCR, gel electrophoresis, and fluorescence microscopy.
Single-Drop Fluorimeter
This device detects DNA based off of fluorescence from a molecule bound to the DNA such as SYBR green dye. The DNA sample that is placed on the slide which is then inserted in the machine and shined with blue LED light. After the light hits the sample, the DNA should emit a dim green glow. To collect data a phone is used to capture the green light.
How the Fluorescence Technique Works
Fluorescence technique works by adding dye to a concentration of DNA, the dye that was used was SYBR Green Dye. The Teflon coated glass will keep the droplets in place.From the single-drop fluorimeter a beam of blue light is emitted. Also a fluorescent green light will be emitted from the droplet which could then be measured by taking a picture on a smart phone and afterwards using Image J to analyze the image.
Procedure
Smart Phone Camera Settings
Type of Smartphone: Samsung Galaxy S3
Flash: None
ISO setting: 800
White Balance: Auto
Exposure: Set on the highest exposure setting (2.0)
Saturation: Not applicable
Contrast: Not applicable
Type of Smartphone: Motorola Razr HD Maxx
Flash: None
ISO setting: Not applicable
White Balance: Auto
Exposure: Set on the highest exposure setting
Saturation: Not applicable
Contrast: Not applicable
Calibration
A light box was placed over the fluorimeter so that no outside source of light can get in. As seen below, the apparatus must then be set up so that the camera lens of the phone is at a 90 degree angle relative to the droplet. After the image of the droplet on the phone was focused, the distance of the camera lens from the droplet was measured using a ruler.
Distance between the smart phone cradle and drop = 5.5 cm for S3 and 9.0 cm for Razr
Solutions Used for Calibration
Calf Thymus DNA Solution (mg/mL)
Volume of DNA (μL)
Volume of SYBR Green I Dye (μL)
Final DNA Concentration (mg/mL)
0
80
80
blank
0.25
80
80
0.125
0.5
80
80
0.25
1
80
80
0.5
2
80
80
1
5
80
80
2.5
Placing Samples onto the Fluorimeter
A glass slide was properly placed with the smooth side down onto the fluorimeter. The first row of the circles is placed in alignment with the blue light of the fluorimeter.
Taking the micropipette, the volume the micropipette will be measuring should be 80 μL.
80 μL of the SYBR Green dye is placed in the middle of the first row of circles.
80 μL of 0 mg/mL calf thymus DNA solution is placed on the SYBR green dye drop.
With the phone properly placed at the distance indicated by the calibration and set with a 5-second timer, the black box was placed around the system, as seen in the image below.
Pictures were taken with the black box closed to minimize the amount of light entering the system.
Repeat the above steps with each of the calf thymus DNA solutions, moving down a row of circles each time to prevent cross-contamination.
Data Analysis
Representative Images of Samples
Image J Values for All Samples
Calf Thymus DNA Concentration (FINAL), ìg/mL
Image
AREA
Mean Pixel Value
RAWINTDEN OF THE DROP
RAWINTDEN OF THE BACKGROUND
2.5
S3 #1
7296
20.229
147593
596
2.5
S3 #2
5238
24.158
126541
712
2.5
Razr
3544
21.257
75336
378
1
S3 #1
6656
17.198
114468
1726
1
S3 #2
7484
18.79
140624
1019
1
Razr
3954
20.256
80092
3516
0.5
S3 #1
6400
14.445
92450
1932
0.5
S3 #2
6784
16.593
112568
911
0.5
Razr
3644
15.472
56381
794
0.25
S3 #1
6892
10.166
70066
1107
0.25
S3 #2
6840
10.635
72740
1162
0.25
Razr
5507
21.861
120522
9814
0.125
S3 #1
8129
16.839
136886
1428
0.125
S3 #2
6780
13.416
90959
1458
0.125
Razr
314
12.911
4054
402
0
S3 #1
4932
16.067
79241
26636
0
S3 #2
4705
14.977
70465
2123
0
Razr
406
15.099
6130
217
Fitting a Straight Line
Possible Error
While the data points taken using the Samsung Galaxy phone had high precision, the data points taken using the Motorola Razr were found to be most likely inaccurate due to the exceedingly low values when compared to the acceptable range of values. This was due, most likely, to the phone camera's low adjustibility in regards to settings and the inferiority of the sensor when compared to the Samsung Galaxy.