SYBR Green Dye
SYBR green dye is a molecular dye that fluoresces in the presence of double stranded DNA, but fluoresces weaker in the presence of single stranded DNA. The dye stains the dsDNA produced through polymerase chain reaction (PCR).
Single-Drop Fluorimeter
The single-drop fluorimeter device detects and measures fluorescence by identifying and quantifying specific fluorescent molecules in a medium. The quantity of fluorescence is directly related to the amount of fluorescent material as well as the amount of molecule that is being detected. Below is an image of the single-drop fluorimeter device.
How the Fluorescence Technique Works
A droplet of the material is placed on the superhyrophobic slide and is exposed to two light beams that decrease the surrounding noise. The fluorescent molecules generate light through excitation by shining light of a shorter wavelength. The energy is released in the form of a photon; the amount released will determine the photon's wavelength and the color of fluorescence which is specific to the SYBR green dye.
Procedure
Smart Phone Camera Settings
We used an application for the camera and the timer on the iPhone and all of the settings were auto.
Calibration
Distance between the smart phone cradle and drop = 10 cm
Distance between the smart phone camera to drop = 9 cm
[Instructions: See worksheet page 6.]
The picture above shows the distance at which the smart phone was placed from the fluorimeter.
The image above shows the entire setup under the black box while the samples were being photographed.
Calf Thymus DNA Solution concentration (µg/mL)
Volume of the 2X DNA solution (µL)
Volume of the SYBR Green
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
To be ensure the samples placed on the slide would result in the correct calibration, the fluorimeter had to be set up properly. The slide was placed onto the fluorimeter and adjusted so that the line of blue light fell directly in between the first two rows of dots. After setting up the fluorimeter, the micro-pipetter was calibrated to 80 microliters and then attached to a clean tip. The tip was inserted into the SYBR Green microtube and then transferred to the slide in between the first two dots so that the blue light illuminated the drop. The contaminated tip was then thrown away. Using a fresh tip still at 80 microliters, the 0 concentration calf thymus was transferred to the slide on top of the already existing SYBR Green drop. The tip contaminated with calf thymus was also thrown away. After aligning the camera on the same plane as the slide and the blue light was directly in line with the drop, a black box was put over the fluorimeter and camera to ensure the blue light was the only light source. The micropipetter then transferred the drop on the slide into the red Dixie cup for proper waste disposal. This process was repeated five times with 0.25, 0.5, 1, 2, and 5 concentrations respectively. Each time, however, the positioning of the drops changed each time, using the next pair of dots with the blue light instead of just sticking with the first two rows the whole time.
Data Analysis
Representative Images of Samples
This is an image where a circle was drawn around the droplet with the freehand tool for a sample with no DNA
This is an image where a circle was drawn around the droplet with the freehand tool for a sample with DNA
Image J Values for All Samples
Calf Thymus DNA Concentration (FINAL), ìg/mL
'
AREA
Mean Pixel Value
Standard Deviation
Of Drop
RAWINTDEN - BACKGROUND
Subtracted drop & Background
5
image 1
4748
237.792
33.446
1129037
10461
1118576
5
image 2
4154
242.575
28.777
1007657
9035
998622
5
image 3
4868
236.993
37.511
1153682
11881
1141801
2
image 1
11985
205.929
67.102
2468057
18891
2449166
2
image 2
13688
197.781
68.269
2707229
21565
2685664
2
image 3
12756
199.634
67.639
2546535
19279
2527256
1
image 1
5265
176.592
67.582
929758
5882
923876
1
image 2
3852
128.091
66.473
493406
5122
488284
1
image 3
5778
214.353
48.057
1238529
6692
1231837
0.5
image 1
13329
108.699
58.687
1448846
18442
1430404
0.5
image 2
13674
121.774
52.412
1665140
15932
1649208
0.5
image 3
12059
119.689
52.823
1443330
22226
1421104
0.25
image 1
4816
52.223
63.274
251508
5646
245862
0.25
image 2
5052
32.584
49.664
164615
6841
157774
0.25
image 3
5336
39.38
58.745
210133
6694
203439
0
image 1
13845
52.334
71.433
724565
14662
709903
0
image 2
11992
66.351
75.566
795687
12082
783605
0
image 3
13088
44.895
62.211
587588
23493
564095
Fitting a Straight Line
The graph above shows the DNA concentration of the drops for every data point. There were three different data points per concentration.
As we were analyzing our data, we found out that our pictures were not calibrated correctly. Therefore, we decided to calibrate all of the data by hand on our graph. Once we had our data values such as the standard deviation, we multiplied the standard deviation by 2 because we wanted to find our error bar range. The averages and the error bars are shown in the graph above.
BME 100 Fall 2013
