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
[Instructions: See worksheet page 6.]
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
[Add more rows as needed]
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
[Instructions: Show an IMAGE where you drew a circle around the droplet with the freehand tool for a sample with no DNA][Instructions: Show an IMAGE where you drew a circle around the droplet with the freehand tool for a sample with DNA (positive signal)]Image;2.5 oval.png
Image J Values for All Samples
[Instructions: See worksheet page 8. To save time on typing a new Wiki table from scratch, use THIS TOOL to auto-generate a Wiki table: Excel-to-Wiki Converter. Copy the headers and values from the Excel spreadsheet you made, paste them into the form field, click submit, copy the Wiki code that the tool generated, and replace TABLE GOES HERE (below) with your auto-generated code.]
PCR Product TUBE LABEL
AVERAGE INTDENS VALUE
PCR Product Concentration, µg /mL
Corrected PCR Product Concentration, µg /mL
1. P
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. N
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
TABLE GOES HERE
Fitting a Straight Line
[Instructions: Place an IMAGE of your Excel plot with a line of best fit here. See worksheet page 9]
Below is the graph illustrating the corrected concentrations compared to the intden values found through imagej.
BME 100 Spring 2014
