OUR TEAM
LAB 5 WRITE-UP
SYBR Green Dye
The SYBR green dye I is used as a stain for nucleic acid testing. SYBR green dye I binds to double stranded DNA and is very fluorescent when bound to double stranded DNA. SYBR Green I absorbs blue light, and emits green light. That is why the blue LED light is used to visualize the fluorescence. Furthermore, SYBR Green Dye I also emits fluorescence when binding to PCR products that contain double-stranded DNA, or dsDNA. However, the SYBR green dye I does not illuminate as well with water or single stranded DNA, which makes it a highly useful product when measuring the amount of PCR product that was created.
Single-Drop Fluorimeter
This instrument detects fluorescence, which is proportional to the amount of fluorescent material, and the amount of the molecule that is detected. The single drop fluorimeter works by measuring the intensity of the excitation of the light, and the wavelength distribution of this light throughout the drop. Optical caustics is a key idea that is applicable in the use of the single drop fluorimeter.
How the Fluorescence Technique Works
The surface of the slide is covered with extremely water-resistant Teflon-- however, the slide also has separate sections which are simply glass. This allows for the blue LED light pay special focus to the droplet, which should increase the intensity and the degree to which fluorescence can be detected. The difference in the surface of the slide allows for the SYBR green dye to bind with DNA and form a substance that ends up going closer to the surface. The use of the lightbox makes sure that it is easier to detect fluorescence within the drop. This is repeated for each calibration solution, which ranges in the concentration of DNA from 0 mg/mL to 2mg/mL. The images from each of these solutions is analyzed by ImageJ, to find the RAW INTDEN of both the droplet and the background.
Procedure
Smart Phone Camera Settings
- Type of Smartphone: iPhone4s
- Flash:none.
- ISO setting: 800
- White Balance: Auto
- Exposure:N/A
- Saturation: N/A
- Contrast: N/A
Calibration
- Distance between the smart phone cradle and drop = 6cm
Solutions Used for Calibration
| Calf Thymus DNA solution concentration (microg/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
- Put a glass slide into the fluorimeter, making sure that the light is in-between two rows.
- Place a 80 microliter drop of SYBER Green in the middle of the first two rows using the pipette. (Make sure it looks like a ball)
- Next add 80 microliters of the calibration solutions listed.
- Align a camera at the edge of the slide to take an image of the drop.
- Place a timer on the camera and take 3 images, immediately close the lid to ensure that little to no light enters the box.
- Use the micropipette to remove the drop and dispose of it and the tip.
Data Analysis
Representative Images of Samples
This is the image of the droplet without DNA in it. 
This is the image of the droplet with DNA in it.
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
|
| 2.5 |
image 1 |
116820 |
115.611 |
13505698 |
585970
|
| 2.5 |
image 2 |
117868 |
113.553 |
13384211 |
456043
|
| 2.5 |
image 3 |
1084 |
114.81 |
124454 |
6409
|
| 1 |
image 1 |
143848 |
101.92 |
14661050 |
376378
|
| 1 |
image 2 |
129212 |
106.759 |
13794585 |
1563279
|
| 1 |
image 3 |
1060 |
116.125 |
123093 |
9701
|
| 0.5 |
image 1 |
1276 |
73.624 |
93944 |
4321
|
| 0.5 |
image 2 |
1292 |
76.67 |
99058 |
4763
|
| 0.5 |
image 3 |
1328 |
72.082 |
95725 |
5579
|
| 0.25 |
image 1 |
1857 |
26.037 |
48351 |
1496
|
| 0.25 |
image 2 |
1866 |
28.88 |
53891 |
1788
|
| 0.25 |
image 3 |
2058 |
24.375 |
50163 |
2002
|
| 0.125 |
image 1 |
163132 |
12.138 |
19800788 |
10506
|
| 0.125 |
image 2 |
156588 |
8.62 |
1349719 |
68339
|
| 0.125 |
image 3 |
146880 |
6.912 |
11888 |
1809
|
| 0 |
image 1 |
165796 |
16.31 |
2704074 |
7666
|
| 0 |
image 2 |
173668 |
7.418 |
1288196 |
15942
|
| 0 |
image 3 |
175244 |
7.8 |
1366883 |
6792
|
|
|
Fitting a Straight Line
|
BME 100 Fall 2013
