OUR TEAM
LAB 5 WRITE-UP
Background Information
SYBR Green Dye
SYBR green dye is used in molecular biology as a nucleic acid stain. The dye generally binds to double-stranded DNA, while staining single-stranded DNA. The DNA-dye-complex that forms emits a green light (wavelength of approx. 520 nm) and absorbs blue light (wavelength of approx. 497 nm). SYBR green dye is commonly used in various methods of quantitative PCR.
Single-Drop Fluorimeter
A device that measures the fluoresence within a given drop of DNA solution. The quantity of fluorescence is generally directly related to the amount of florescent material in the given solution. The fluorimeter is capable of measuring the intensity and wavelength distribution of the spectrum that is emitted when the light passes through the DNA solution.
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How the Fluorescence Technique Works
The fluorescence technique works by using a dye that makes the proteins glow a fluorescent green, showing whether the protein is either present or is not.
Procedure
Smart Phone Camera Settings
- Model: HTC One
- Flash: No
- ISO setting: 100
- White Balance: Fluorescent
- Exposure: 0 (Default)
- Saturation: 0 (Default)
- Contrast: 0 (Default)
Calibration
We set the light and slides about 7 cm from the smartphone camera and we set the box over it to block out the light.
- Distance between the smart phone cradle and drop = 7cm
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Solutions Used for Calibration
| Calf Thmus Dna solution concentration (microg/mL) |
Volume of the 2X DNA solution (microliters) |
Volume of the SYBR GREEN I Dye solution (microliters) |
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
|
| 0.125 |
80 |
80 |
blank
|
Placing Samples onto the Fluorimeter
- Place the slide in its designated place, smooth side down, into the single droplet Fluorometer device.
- Micro-pipette 80µL of the SYBR green dye and place it in the second row of the middle column of the slide.
- Next carefully Micro-pipette 80µL of desired concentration of calf thymus DNA on top of the SYBER green dye onto the slide.
- The total amount of SYBR green dye/ calf thymus DNA mixture will be 160µL
- Start test by taking a photo of the droplet.
Data Analysis
Representative Images of Samples
No DNA
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With DNA
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Image J Values for All Samples
| Calf Thymus DNA Concentration (FINAL), μg/mL
|
Image Number
|
AREA
|
Mean Pixel Value
|
RAWINTDEN OF THE DROP
|
RAWINTDEN OF THE BACKGROUND
|
| 2.5 |
1 |
33691 |
132.209 |
4454265 |
4454265
|
| 2.5 |
2 |
33840 |
131.203 |
4439919 |
4439919
|
| 1 |
1 |
36244 |
79.188 |
2870091 |
2870091
|
| 1 |
2 |
43552 |
79.813 |
3476030 |
3476030
|
| 0.5 |
1 |
37294 |
37.722 |
1406812 |
1406812
|
| 0.5 |
2 |
31940 |
38.819 |
1239877 |
1239877
|
| 0.25 |
1 |
43038 |
23.1 |
994189 |
994189
|
| 0.25 |
2 |
35870 |
27.141 |
973557 |
973557
|
| 0.125 |
1 |
41800 |
6.658 |
278307 |
278307
|
| 0.125 |
2 |
39159 |
5.107 |
199977 |
199977
|
| 0 |
1 |
44228 |
6.221 |
275159 |
275159
|
| 0 |
2 |
39651 |
7.252 |
287535 |
287535
|
Note: Only two images needed per Dr. Garcia since there were initial problems with the HD camera
Fitting a Straight Line
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|
BME 100 Fall 2013
