SYBR Green Dye
SYBR Green I is a fluorescent dye that binds to double stranded DNA. This dye then fluoresces green when excited by blue light. SYBR green dye is very efficient at visualizing DNA, incredibly safe to users, and cheap, making it a prime dye for basic DNA displaying.
Single-Drop Fluorimeter
A fluorimeter is a device used to measure amounts of fluorescence. The device usually measures fixed wavelengths either by filters, monochromators or fixed diodes. Fluorimeters have two light detectors: one light detects the absorbance, while the other detects fluorescence emission.
Drops of DNA solution on flourimeter slide
How the Fluorescence Technique Works
The fluorescence technique measures the concentration of DNA in mixed liquids by adding a fluorescent dye and measuring the amount of fluorescence. This is done using a single drop fluorometer with a Teflon-coated glass slide. The slide's surface is not completely covered; circles of bare glass remain, which water sticks to, allowing drops of liquids to stay in place. Drops of DNA solution are placed on the glass circles along with SYBR Green I. Blue LED light from the fluorometer is aimed at the drops, exciting the dye and causing it to fluoresce. This fluorescence is then analyzed to determine the concentration of DNA in the solution.
Procedure
Smart Phone Camera Settings
Iphone 4
Flash: Off
ISO setting: Auto
White Balance: Auto
Exposure: Auto
Saturation: Auto
Contrast: Auto
Calibration
Place the smartphone on the cradle at a right angle
Fix the height of the fluorimeter so that the camera can take picture of the drop at a right angle
Distance between the smartphone cradle and drop = 4cm
Solutions Used for Calibration
Calf Thymus DNA solution concentration (μg/mL)
Volume of the 2x DNA solution (μL)
Volume of the SYBR GREEN Dye Solution (μL)
Final DNA concentration PicoGreen 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
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Placing Samples onto the Fluorimeter
Step one: Check your phone camera setting, and turn the flash off
Step two: Pick up DNA, gloves, lab coat, glass slide, buffer tubes, SYBR Green I tube, and DNA standard
Step three: Slide the glass slide into fluorimeter
Step four: Drop 80μL of the solution where the light is flashing
Step five: Put the box around it and take pictures of the drops
Data Analysis
Representative Images of Samples
Circle analysis of DNA negative drop (0μg/mL)
Circle Analysis of DNA positive drop (5μg/mL)
As shown, the DNA positive drop is much brighter.
Image J Values for All Samples
Trial
DNA Concentration (μg/mL)
Area
Mean Pixel Value
RAWINTDEN (drop)
RAWINTDEN (background)
1
0
10,906
21.672
236,351
70,338
1
0.25
10,044
35.971
361,290
42,572
1
0.5
10,562
84.274
890,103
52,974
1
0.5
10,452
50.546
528,303
50,012
1
1
10,350
115.226
1,192,594
39,995
1
2
10,914
193.145
2,107,989
46,923
1
5
10,282
142.563
1,465,554
55,342
2
0
10,808
50.412
544,856
71,106
2
0.25
10,510
51.038
536,412
39,795
2
0.5
10,688
78.799
842,203
57,075
2
1
10,432
90.965
948,944
52,390
2
2
10,103
152.075
1,536,481
61,000
2
5
10,440
115.674
1,207,635
51,873
3
0
10,131
37.264
377,520
66,289
3
0.25
10,448
61.987
647,645
48,887
3
0.5
10,762
64.475
693,875
40,978
3
1
10,465
74.835
783,144
47,549
3
2
10,188
155.63
1,585,554
43,186
3
5
10,672
72.312
771,711
47,142
Fitting a Straight Line [Place an IMAGE of your Excel plot with a line of best fit here. See worksheet page 8]
BME 103 Spring 2013
