OUR TEAM
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LAB 2 WRITE-UP
Background Information
SYBR Green Dye
[A short summary describing SYBR green dye]
SYBR Green I is a fluorescent dye that is effectively used to bind double stranded DNA.
Single-Drop Fluorimeter
[A description of the single-drop fluorimeter device. Add a PHOTO for bonus points]
Fluorimeter is a device that is used to measure fluorescence.
How the Fluorescence Technique Works
[In your own words, a summary of the information from page 9 of the worksheet]
Procedure
Smart Phone Camera Settings
- Iphone 4
- Flash: Off
- ISO setting:NA
- White Balance: NA
- Exposure: NA
- Saturation: NA
- Contrast: NA
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)
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5 |
80 |
80 |
2.5
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2 |
80 |
80 |
1
|
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 |
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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 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
[Show an IMAGE where you drew a circle around the droplet with the freehand tool for a sample with no DNA]
[Show an IMAGE where you drew a circle around the droplet with the freehand tool for a sample with DNA (positive signal)]
Image J Values for All Samples [See worksheet page 5]
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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[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]
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