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OUR TEAM
LAB 2 WRITE-UP
SYBR Green Dye
SYBR Green I is a fluorescent dye that binds to double-stranded DNA and fluoresces green when excited by blue light. SYBR green dye is very safe to users and cheap, making it ideal for basic DNA visualization.
Single-Drop Fluorimeter
A fluorimeter is a device used to measure amounts of fluorescence. Fluorimeters usually measure fixed wavelengths either with filters, monochromators, or fixed diodes. Fluorimeters have two light detectors; one detects 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 a solution 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 liquid 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 to both the table and the fluorimeter slide
- Adjust the height of the fluorimeter slide if necessary so the camera is level with the drop
- 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 I Dye Solution (μL) |
Final DNA Concentration PicoGreen Assay (ng/mL)
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| 5 |
80 |
80 |
2.5
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| 2 |
80 |
80 |
1
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| 1 |
80 |
80 |
0.5
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| 0.5 |
80 |
80 |
0.25
|
| 0.25 |
80 |
80 |
0.125
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| 0 |
80 |
80 |
blank
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Placing Samples onto the Fluorimeter
- Step one: Adjust camera settings and turn the flash off
- Step two: Obtain calf thymus DNA, gloves, lab coat, glass slide, buffer tubes, and SYBR Green I
- Step three: Insert Teflon-coated glass slide into fluorimeter
- Step four: Drop 80μL of solution onto slide and position in front of blue light
- Step five: Put dark box over fluorimeter and smartphone and take pictures of the drops
Data Analysis
Representative Images of Samples
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Circle analysis of DNA negative drop (0μg/mL)
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Circle Analysis of DNA positive drop (5μg/mL)
As shown, the DNA positive drop is much brighter than the DNA negative drop.
Image J Values for All Samples
| Trial |
DNA Concentration (μg/mL) |
Area |
Mean Pixel Value |
RAWINTDEN (drop) |
RAWINTDEN (background)
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| 1 |
0 |
10,906 |
21.672 |
236,351 |
70,338
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| 1 |
0.25 |
10,044 |
35.971 |
361,290 |
42,572
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| 1 |
0.5 |
10,562 |
84.274 |
890,103 |
52,974
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| 1 |
0.5 |
10,452 |
50.546 |
528,303 |
50,012
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| 1 |
1 |
10,350 |
115.226 |
1,192,594 |
39,995
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| 1 |
2 |
10,914 |
193.145 |
2,107,989 |
46,923
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| 1 |
5 |
10,282 |
142.563 |
1,465,554 |
55,342
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| 2 |
0 |
10,808 |
50.412 |
544,856 |
71,106
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| 2 |
0.25 |
10,510 |
51.038 |
536,412 |
39,795
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| 2 |
0.5 |
10,688 |
78.799 |
842,203 |
57,075
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| 2 |
1 |
10,432 |
90.965 |
948,944 |
52,390
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| 2 |
2 |
10,103 |
152.075 |
1,536,481 |
61,000
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| 2 |
5 |
10,440 |
115.674 |
1,207,635 |
51,873
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| 3 |
0 |
10,131 |
37.264 |
377,520 |
66,289
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| 3 |
0.25 |
10,448 |
61.987 |
647,645 |
48,887
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| 3 |
0.5 |
10,762 |
64.475 |
693,875 |
40,978
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| 3 |
1 |
10,465 |
74.835 |
783,144 |
47,549
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| 3 |
2 |
10,188 |
155.63 |
1,585,554 |
43,186
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| 3 |
5 |
10,672 |
72.312 |
771,711 |
47,142
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Fitting a Straight Line
General trendline of INTDENS readings vs. DNA concentration
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BME 103 Spring 2013
