SYBR Green dye is a small molecular dye that fluoresces in the presence of DNA but fluoresces weakly in the presence of water or single strands of DNA. The green dye detects double-stranded DNA generated during PCR. It is also used to detect DNA in gel electrophoresis.
Single-Drop Fluorimeter
The Single-Drop Fluorimeter is a device used to measure fluorescence. The base of the fluorimeter houses a slide holder specialized to hold a rectangular glass slide that has a hydrophobic coating. This coating allows water to collect in a droplet on the surface that will be used to measure the fluorimeter's light source, a blue LED light. The base also allows a camera to sit in front of the slide. Finally, a light box is placed over the base that encloses the fluorimeter and prevents light from outside sources from entering, making the droplet and LED more visible for the camera to capture.
How the Fluorescence Technique Works
Fluorescence is a process in which the emission of light from a sample droplet is measured in order to collect data. For this experiment specifically, a blue LED is passed through a droplet containing a sample of DNA and SYBR Green dye which will fluoresce when interacted together. This LED light excites the molecules within the droplet causing the droplet to emit light in a more focused wavelength which can be measured visibly when captured in a picture using a camera. On a more theoretical level, the light given off by the droplet is measured by the equation of fluorescence, which states that the product of the intensity of the LED, the absorption coefficient and molar concentration of SYBR Green dye, and a value of quantum efficiency, equals the fluorescent intensity. This fluorescent intensity is then related to the concentration of DNA in the droplet when removing the background fluorescence of non-reacting molecules (Week10 Lab Handout-BME 100).
Procedure
Smart Phone Camera Settings
Type of Smartphone: iPhone 5s
Flash: off
ISO setting: n/a
White Balance: n/a
Exposure: n/a
Saturation: n/a
Contrast: n/a
Calibration
Distance between the smart phone cradle and drop = 5 cm
Solutions Used for Calibration
Calf Thymus DNA (μg/mL)
SYBR Green Dye Volume (μL)
2X DNA Solution Volume (μL)
Final DNA Concentration (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
Step One: Insert a glass slide into the fluorimeter with the "smooth side" facing down.
Step Two: Extract X ml of SYBR green fluid and place it on the sample slide between the First and Second row on the middle dots.
Step Three: Extract and place X ml of the sample concentration that is to be tested on the same fluid that was used int he previous step.
Step Four: After each photo has been taken, remove the sample fluid and dispose of it as instructed.
Step Five: Move the slide into position in such that the blue light is directed onto the middle of the next 2 dots that have not been used.
Step Six: Repeat steps 1-5 until all samples are tested. Remember to use a new slide when each dot has been used on the slide.
Data Analysis
Representative Images of Samples
Positive Signal
Image J Values for All Samples
Calf Thymus DNA Concentration (FINAL), μg/mL
'
AREA
Mean Pixel Value
RAWINTDEN OF THE DROP
RAWINTDEN OF THE BACKGROUND
RAWINTDEN - BACKGROUND
2.5
image 1
57175
91.476
5017524
101447
4916077
2.5
image 2
56606
89.232
5051060
158329
4892731
2.5
image 3
57980
94.259
5465111
93588
5371523
1
image 1
54792
92.851
5087471
107397
4980074
1
image 2
58002
77.445
4491953
90963
4400990
1
image 3
53835
74.786
4026096
92974
3933122
0.5
image 1
51093
77.155
3942062
82723
3859339
0.5
image 2
49822
83.888
4179470
75890
4103580
0.5
image 3
52412
86.055
4510312
90407
4419905
0.25
image 1
49036
85.196
4177657
80663
4096994
0.25
image 2
54926
88.903
4883089
95912
4787177
0.25
image 3
49940
78.76
3933264
93143
3840121
0.125
image 1
46861
54.974
2576142
88321
2487821
0.125
image 2
51066
53.409
3238055
89218
3148837
0.125
image 3
56664
87.337
4948882
100275
4848607
0
image 1
47032
46.163
2171138
76774
2094364
0
image 2
51824
49.35
2557534
77391
2480143
0
image 3
46976
57.165
2685374
178179
2507195
Fitting a Straight Line
Disclaimer: It is also worth noting that the RAWINTDEN values are much lower than the theoretical values. In addition, there is less than optimal change of the RAWINTDEN values between the concentrations of the DNA. Even though the difference is minute, it is still measurable, and these data points can still be used for a calibration as long they are consistent with the values in the next experiment using the actual PCR DNA.
BME 100 Fall 2013
