Name: Antonio Moran Role: Took pictures and worked on Procedure section.
Name: Brandon Bartels Role: Worked on procedure section and took a couple of the photos.
Name: Robel Yoseph Role: Background Information and Photographer
LAB 5 WRITE-UP
Background Information
SYBR Green Dye
SYBR green dye is a small molecular dye that is used to stain nucleic acids. It fluoresces very well in the presence of double stranded DNA but fluoresces very weakly in water or with single stranded DNA, and is used to detect polymerase chain reactions. During a polymerase chain reaction, SYBR green binds to the double stranded DNA and quantifies non-specific and specific PCR products. In order to be used correctly, SYBR green dye must be calibrated before any tests can be made using it. When being used in tests, the dye will bond to DNA strands emit a green light when it is exposed to blue light, but when exposed to white light the effects are washed out and no visible changes can be seen.
Single-Drop Fluorimeter
Single-Drop Fluorimeter
The Single-Drop Fluorimeter is an instrument that detects the amount of fluorescence in test samples. The fluorescence that is measured has a proportional relationship to the amount of florescent material and, less directly, proportional to the amount of the molecules that are being detected. The amount of molecules present is related to the amount of fluorescent material that is there. The device is made up of a small plastic box with a slot on top for slides with samples to be placed upon it. On the right hand side of the device is a switch that controls the LED light. The slot that lies along the top of the device runs the entire length of the box and allows for the blue LED light to shine across the samples on multi-welled slides. In the picture to the left, the switch on the right of the fluorimeter is the power switch. On the top of the fluorimeter is a place to put the slides. And the glowing blue LED light to the right of the slide is what illuminates through the sample.
How the Fluorescence Technique Works
First, place the slide with the smooth side down into the fluorimeter slot. Adjust the height in order to make sure the camera positioning is right. Place 80 uL of the SYBR green dye between the two circles on the slide. Then add 80 uL of the DNA solution on top of where the green dye was placed. Adjust the slide so that light passes through the middle. Set the timer on the camera until it is focused and then block any external light sources by lowering the flap. Once a picture is taken, it can be uploaded to ImageJ. After everything is done with, the dyes can be disposed of in a proper waste container.
Procedure
Smart Phone Camera Settings
Type of Smartphone: iPhone 4s
Flash: None
ISO setting: Default
White Balance: Default
Exposure: Default
Saturation: Default
Contrast: Default
Type of Smartphone: iPhone 5
Flash: None
ISO setting: Default
White Balance: Default
Exposure: Default
Saturation: Default
Contrast: Default
Calibration:
The fluorimeter was placed on top of three plates so that it was level with the hight of the iphone's camera. This was because the phone needed to be able to take a picture of the side of the drop. The phone was placed on a stand in front of the fluorimeter and the camera on the phone was about 7 cm away from the drop. This was because the camera needed to be at least 4 cm away from the fluorimeter in order to prevent a blurry photo, and we wern't able to get the camera any closer to the fluorimeter because of the necessary stands underneith the fluorimeter.
Distance between the smart phone cradle and drop = 7 cm
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 in SYBR Green I 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
Take 80uL of SYBR Green I solution and place it on the first two clear circles in the middle of the slide.
Discard tip.
Take 80uL of sample and add it to the drop. It should now contain 160uL, and the sample will be diluted by half.
Discard Tip.
Place slide on the fluorimeter.
Take Photo.
To place another drop on the slide, first remove and discard the 160uL of diluted sample, and discard tip.
Then repeat the first steps, but on the second two clear circles in the middle of the slide.
Data Analysis
Image of drop with DNA: positive signal (left picture)
Image of drop without DNA: negative signal (right picture)
Calf Thymus DNA Concentration (FINAL), μg/mL Robel
'
AREA
Mean Pixel Value
RAWINTDEN OF THE DROP
RAWINTDEN OF THE BACKGROUND
2.5
image 1
17148
85.963
1474099
44312
2.5
image 2
14596
96.033
1401700
30629
2.5
image 3
16380
87.129
1427177
45502
1
image 1
28428
76.255
2167784
201558
1
image 2
31140
76.532
2383215
86646
1
image 3
32450
64.967
2108191
96341
0.5
image 1
27960
49.391
1380983
67206
0.5
image 2
32308
58.034
1874952
116477
0.5
image 3
31289
68.453
2141825
149224
0.25
image 1
31316
55.766
1746363
76336
0.25
image 2
29292
56.421
1652697
70746
0.25
image 3
26800
60.862
1631094
72910
0.125
image 1
1860
29.625
553404
143432
0.125
image 2
20084
34.631
695536
98156
0.125
image 3
21392
28.595
611711
50055
0
image 1
2163
13.675
29579
11295
0
image 2
3024
13.947
30042
12432
0
image 3
2589
12.056
31003
11982
Calf Thymus DNA Concentration (FINAL), μg/mL LT
'
AREA
Mean Pixel Value
RAWINTDEN OF THE DROP
RAWINTDEN OF THE BACKGROUND
2.5
image 1
3213
32.265
119732
10223
2.5
image 2
2988
36.342
108590
9800
2.5
image 3
1752
20.894
176767
6312
1
image 1
2510
43.255
108571
9005
1
image 2
1904
50.73
100648
6899
1
image 3
2541
44.605
113341
9387
0.5
image 1
2278
44.776
101999
10902
0.5
image 2
2648
45.679
120959
12005
0.5
image 3
2799
46.907
131293
13477
0.25
image 1
2489
58.927
146670
13081
0.25
image 2
2873
64.187
184409
13731
0.25
image 3
2934
62.008
181931
13722
0.125
image 1
2320
15.488
35932
11337
0.125
image 2
1987
15.38
30561
10794
0.125
image 3
1892
16.489
31197
9357
0
image 1
2034
12.473
15760
10345
0
image 2
2546
14.032
31002
115587
0
image 3
2143
12.591
24902
19543
Faulty SYBR Green Dye may have caused the results to be abnormal.
BME 100 Fall 2013
