SYBR Green Dye
SYBR Green dye is a cyanine dye that is used in biology. It is used as a nucleic acid stain that binds to double stranded DNA. It can also be used to view single stranded DNA and RNA but with lower performance. The dye can be used to stain DNA in PCR reactions.
Single-Drop Fluorimeter
The single drop fluorometer is used to measure the amount of fluorescence in each sample by emitting a blue LED light. It is a small box, and the slides with the samples go on the top. The LED light can shine across the slides to light up the samples.
There are two pictures of the fluorometer. The one on the right shows the set up, with the device, and the stand holding the phone/camera upright. The photo on the left shows the shade over the device which is what is would look like while actually performing.
How the Fluorescence Technique Works
Start with the smooth side of the slide down, and place it at the height of the camera. Add 80 uL of dye and 80 uL of DNA between the circles on the slide. The LED light will focus on the sample and the dye will bind with the DNA. The timer needs to be set on the camera to allow time to close the shade and block out any light. Analyzing the photos will show the amount of fluorescence and the concentration of the sample.
Procedure
Smart Phone Camera Settings
Type of Smartphone: HTC EVO 4G LTE
We weren't able to adjust the settings of the camera on the smartphone. No flash was used. The ISO, white balance, exposure, saturation and contrast were all on the auto setting.
Type of Smartphone: LG Spectrum 2
We weren't able to adjust the settings of the camera on the smartphone. No flash was used. The ISO, white balance, exposure, saturation and contrast were all on the auto setting.
Calibration
First, to calibrate the set up the excitation light, the blue LED, is switched on. The camera is turned on and placed in the cradle that holds the phone upright at a decent angle and height for viewing the sample. The flourimeter and the sample is adjusted vertically through the use of plastic trays. This is done to ensure the photo of the drop would be of the sample from the correct viewing angle. The distance of the cradle from the sample is adjusted in order to be as close as possible without blurring of the image.
The distance between the smart phone cradle and drop was .4 centimeters.
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 in the fluorimeter with the rough hydrophobic side facing up.
Use a micropipette to obtain 80 µL of SYBR Green I solution and dispense the drop on the first two clear circles in the middle of the slide.
With a clean tip, use the micropipette to measure and obtain 80 µL of the sample solution. Dispense the drop in the same position and on top of the SYBR Green I drop.
Position the slide in the fluorimeter so that the light goes through the center of the drop.
Place a smartphone in the cradle at a distance that is less than 4 cm so that a side view is obtained when an image is taken.
Place the lightbox over the device and set the phone timer to take an image of the slide.
Data Analysis
Representative Images of Samples
Screenshot of a sample with no DNA:
Screenshot of a sample with DNA:
Image J Values for All Samples
DNA concentration
area
mean pixel value
RAWINTDEN OF THE DROP
RAWINTDEN OF THE BACKGROUND
0
137764
47.709
6572538
47374
0.25
71900
21.916
1575730
10917
0.5
133928
62.233
8334693
17069
1
114176
86.578
10154005
26149
2
50900
114.32
13052648
35108
5
50900
104.588
5323542
35606
0
159348
29.5
4701724
103184
0.25
164859
31.534
5103921
53714
0.5
195754
69.125
13531488
219064
1
153040
77.054
11792353
107546
2
171008
105.575
18054220
95208
5
73048
108.262
7908290
39732
Fitting a Straight Line
Data from one of the 2.5 µg/mL sample and both of the 5 µg/mL samples were ignored as the procedures were done incorectly and including them would result in improper calibration.
BME 100 Fall 2013
