SYBR Green I is a fluorescent dye that is effectively used to bind double stranded DNA. This dye then emits a green light. SYBR green dye is commonly used because it is so efficient at visualizing DNA and is incredibly safe to users. Not to mention large quantities of the dye are cheap, making it a prime dye for basic DNA displaying.
Single-Drop Fluorimeter
Fluorimeter is a device that is used to measure parameters of fluorescence concentration. The device usually measures fixed wavelengths either by filters, monochromators or fixed diodes. The fluorometer have two light detectors. One light detects the absorbance, while the other detects fluorescence emission.
How the Fluorescence Technique Works
Fluorescence Technique is a technique to measure concentration of mixing liquids. This is done by using a single drop fluorometer that contain a slide coated with a layer of Teflon. The slide also contain circles of bare glass, allowing drops of liquids to stay in place. Blue LED light from the fluorometer points to the drops and increase intensity of the fluorescence. SYBR Green I was later added with a solvent to help form the complex DNA.
Procedure
Smart Phone Camera Settings
Iphone 4
Flash: Off
ISO setting: Auto
White Balance: Auto
Exposure: Auto
Saturation: Auto
Contrast: Auto
Calibration
Photo of Fluorimeter
Put your phone on the cradle at a right angle
Then fix the height of the fluorimeter so that the camera can take picture of the drop on sideways
Distance between the smart phone cradle and drop = 4cm
Solutions Used for Calibration
Calf Thymus DNA solution concentration (microg/ML)
Volume of the 2x DNA solution (uL)
Volume of the SYBR GREEN Dye Solution (uL)
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
blank
Placing Samples onto the Fluorimeter
Step one, Check your phone camera setting, and turn the flash off
Step two,Pick up DAN, gloves, lab coat, glass slide, buffer tubes, sync green I tube, and DNA standard
Step three, Slide the glass slide into fluorimeter
Step four, Drop 80 micro liters of sample 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. 0 concentration.
Circle Analysis of DNA positive drop. 5 concentration.
As shown, the DNA positive drop is much more illuminated.
Image J Values for All Samples[See worksheet page 5]
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
[Add more rows as needed]
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
