SYBR Green Dye A fluorescent DNA dye that binds all dsDNA and is measured by the change in fluorescence throughout. The dye is used in the PCR reaction in order to detect double stranded DNA. This allows experimenters to quantify the amount of DNA being produced.
Single-Drop Fluorimeter An instrumentation that is able to detect fluorescence. The quantity is proportional to the number of molecules being deteced.
How the Fluorescence Technique Works A technique that detects light in a molecule much similar to a spectrometer. It uses an ultraviolet light and excites the electrons causing the molecules to emit light.
Procedure
Smart Phone Camera Settings
First Phone:
Type of Smartphone: iPhone 5s
Flash: Off
ISO setting: 800
White Balance: Auto
Exposure: Highest Setting
Saturation: Highest Settings
Contrast: Lowest Settings
Additional Phone:
Type of Smartphone: Galaxy Samsung S3
Flash: Off
ISO setting: 800
White Balance: Auto
Exposure: Highest Setting
Saturation: Highest Setting
Contrast: Lowest Setting
Calibration
First turn on the Blue LED switch in order to activate the excitation light. Also make sure the camera is open with the settings listed above. From there place the pone on the cradle with 90 degree angle from the slide. Make sure to adjust the height of phone and/or fluorimeter in order to take pictures of the drops. Lastly, the cradle should be as close to the fluorimeter as possible with having a clear picture still.
Distance between the smart phone cradle and drop =
4.5 cm
Solutions Used for Calibration
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
On the first two rows of the slide, place one drop (the pipette is calibrated to 80 microlitres) of SYBR Green.
Add a second drop of the diluted calf thymus DNA on top of the first drop on the slide. We now have a sample!
Adjust the slide such that the blue LED light is using the drop to focus on the other side of the drop.
Use the camera's timer to take a picture after closing the light box (this prevents as much outside light as possible from contaminating our data).
Remove the box, being careful not to knock the smartphone out of position.
Use the pipette to remove the entire 160 microlitre drop from the slide, then move the slide into the next position and use the next concentration standard.
Repeat as needed.
Data Analysis
Representative Images of Samples
Negative Signal
Positive Signal
Image J Values for All Samples
PCR Product TUBE LABEL
Diluted Volume (µL)
SYBR GREEN Volume (µL)
INTDEN 1
INTDEN2
INTDEN3
AVERAGE INTDENS VALUE
A1
80
80
2716687
3229970
3340085
3095581
A2
80
80
1451878
2568844
8446955
4155892
A3
80
80
3862714
8011744
1600043
4491500
B1
80
80
9856858
5058003
3108259
6007707
B2
80
80
6862644
5685363
6783718
6443908
B3
80
80
6287959
7119245
9598254
7668486
+
80
80
11779932
7973600
11270538
10341357
-
80
80
4371681
2482949
1977785
2944138
Fitting a Straight Line
Linear Regression of PCR Results
Interpretation
Based on the results from the image analysis, the intensity in the green coloration in patient B was higher than patient A. Due to the fact that false positives and negatives were controlled for, it can be confidently said that patient B's DNA indicated a positive result for gene detection test. Patient A did not test positive. According to the calibration, it should be noted that the concentration of DNA is positively correlated to the green intensity of the SYBR Green dye; thus the high intensity in patient B's amplified DNA would be expected to increase with increased concentration of DNA.
BME 100 Spring 2014
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