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OUR TEAM
LAB 5 WRITE-UP
Background Information
SYBR Green Dye
An asymmetrical cyanine dye that stains the nucleic acids of DNA. The dye binds to the DNA, turning it green in order to detect whether or not it's a DNA flourimeter. If a sample does not turn green in the flourimeter, then it does not contain DNA.
Single-Drop Fluorimeter
A black box device used to measure florescence parameters and patterns. Using the 2 blue LED lights, the Single-Drop Fluorimeter beams through a sample to record data. The data is than converted into an electrical signal and transmitted to a computer
How the Fluorescence Technique Works
The DNA drop sample slides are placed through into the Flourimeter. If properly placed, the light will illuminate the center of the drop which will focus the LED light as it passes through. The pass through magnifies the glow of the DNA which will produce better results. The green color of the SYBR Green Dye is more easily seen at the top of the drop and is easily analyzed. The presence of a green light determines if the sample contains DNA.
Procedure
Smart Phone Camera Settings
- Type of Smartphone:Samsung Galaxy S4
- Flash: Auto
- ISO setting:800
- White Balance: Auto
- Exposure:High
- Saturation:High
- Contrast:Low
Calibration
The smartphone was placed in the cradle perpendicular to the Fluorimeter. The cradle was measured at 12.5 cm away from the fluorimeter.
- Distance between the smart phone cradle and drop = 12.5cm
Solutions Used for Calibration [Instructions: See worksheet page 6.]
| Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL) |
Volume of the 2X DNA solution (µL) |
Volume of the SYBR GREEN I
Dye solution (µL)
|
Final DNA concentration in SYBR Green I solution (µg/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 |
0
|
Placing Samples onto the Fluorimeter
- Place 80 microliter drops of SYBR GREEN to the middle of the first two rows of the slide.
- Add 80 microliters of the desired concentration of the calf thymus to the same spot of the SYBR GREEN.
- Align the drop with the blue LED light.
- Cover the fluorimeter and smartphone and take timed photo.
Data Analysis
Representative Images of Samples
Below is an image depicting a sample analyzed using imagej that had a concentration of 0.
This image shows a sample with concentration of 2.5, as is apparent there is a notable difference between the concentration of 0 as the green light is not visible in the picture above.
Image J Values for All Samples
| PCR Product TUBE LABEL
|
AVERAGE INTDENS VALUE
|
PCR Product Concentration, µg /mL
|
Corrected PCR Product Concentration, µg /mL
|
| 1. Positive |
14109839 |
1.210984 |
0.100915325
|
| 2. A1 |
5644840 |
0.364484 |
0.030373667
|
| 3. A2 |
6512415 |
0.451242 |
0.037603458
|
| 4. A3 |
5443461 |
0.344346 |
0.028695508
|
| 5. Negative |
6025545 |
0.402555 |
0.033546208
|
| 6. B1 |
13694379 |
1.169438 |
0.097453158
|
| 7. B2 |
12199100 |
1.01991 |
0.0849925
|
| 8. B3 |
15195038 |
1.319504 |
0.10995865
|
|
|
The values for concentrations and corrected concentrations can be found above. Note that the positive control possessed a higher concentration of DNA than the negative sample. This is good since those values are used as a threshold for determining if the others are either positive or negative. In the images the negative sample appeared to reflect little to no green at all whereas the positive was very brightly illuminated by the fluorescent light.
Patient ID: 77842 Is listed above using the A values such that the range of concentrations were close to .03. This value and the striking resemblance color-wise to the negative sample leads one to believe that it did not possess the DNA that was tested.
Patient ID:26204 Was labeled using the B values above and was found to have a much higher concentration of the DNA sample. This sample appeared to have been very green when analyzing the images through the analysis software. Given this it makes since to infer that the sample was tested positive for the DNA.
Fitting a Straight Line
Below is the graph illustrating the corrected concentrations compared to the intden values found through imagej.
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BME 100 Spring 2014
