SYBR Green Dye
SYBR green dye is one of the asymmetrical high-sensitivity reagents for staining DNA and RNA in electrophoretic gels. When combined, the gel stains offer greater sensitivity towards measuring an accurate reading. In this lab, specifically, SYBR green dye is primarily used to stain and dye the nucleic acid, which makes it possible to see large amounts of DNA by the naked eye. Essentially, SYBR green dye specifically fluoresces in the actual presence of double stranded DNA. However, in the presence of the single stranded DNA, the SYBR green dye fluoresces even more so. The SYBR Green Dye absorbs blue light and emits green light, explaining the ability to visualize the fluorescence. Generally, SYBR green dye deliver highly sensitive real time PCR results in moderate timings, allowing lab members to process the information in a shorter period of time.
Single-Drop Fluorimeter
The single drop fluorimeter is a device that detects and measures fluorescence by specifying certain fluorescent molecules in a given area. The amount measured is proportional to the amount of fluorescent material to the amount of molecule that is detected. More specifically, the Single-Drop Fluorimeter shines blue light through a drop of DNA/SYBR green dye mix which absorbs the blue light making it possible to see the DNA in the drop of liquid.
How the Fluorescence Technique Works
The Fluorescence Technique Works by using the surface of the slide, which is mainly glass, which allow for the blue LED light to focus specifically on the droplet. Because of the difference in the surface, the SYBR green dye is able to bind with the DNA. Once the data is processed, the images will be used to determine the specificities for each of the solutions. The specific the fluorescent technique works because it gives the person the ability to see DNA with the naked eye so that the proof of DNA is apparent.
Procedure
Smart Phone Camera Settings [Instructions: The type of smart phone you used and how you adjusted the camera settings, if applicable (see worksheet page 4).]
Type of Smartphone: iPhone
Flash: Flash was inactivated.
ISO setting: ISO was set at 800.
White Balance: White Balance was set at auto.
Exposure: Exposure was set to the highest setting.
Saturation: Saturation was set to the highest setting.
Contrast: Contrast was set to the lowest setting.
Calibration
[Instructions: Describe how to set up your camera in front of the fluorimeter. Add a PHOTO of this set-up for bonus points.]
Distance between the smart phone cradle and drop =
[Instructions: See worksheet page 6.]
Solutions Used for Calibration[Instructions: See worksheet page 6.]
Calf Thymus DNA solution concentration (microg/mL)
Volume of the 2X DNA solution (μL)
Volume of the SYBR GREEN I Dye solution (μL)
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
[Add more rows as needed]
Placing Samples onto the Fluorimeter
[Instructions: Step one, in your OWN words]
[Instructions: Step two, in your own words]
[Instructions: Step three, in your own words]
[Instructions: Step etc., in your own words]
Data Analysis
With DNA
Without DNA
Representative Images of Samples
[Instructions: Show an IMAGE where you drew a circle around the droplet with the freehand tool for a sample with no DNA]
[Instructions: Show an IMAGE where you drew a circle around the droplet with the freehand tool for a sample with DNA (positive signal)]
Calf Thymus DNA Concentration (FINAL), μg/mL
'
AREA
Mean Pixel Value
RAWINTDEN OF THE DROP
RAWINTDEN OF THE BACKGROUND
2.5
Image 1
13985
199.45
13236789
12058723
2.5
Image 2
12823
187.45
12789541
11902538
2.5
Image 3
11907
175.34
8923587
110926359
1
Image 1
6627
189.56
13092679
94561092
1
Image 2
7543
192.45
14982673
80268121
1
Image 3
6802
162.91
56823
62390123
0.5
Image 1
13897
218.82
87652
145820372
0.5
Image 2
12096
187.45
99367
18902736
0.5
Image 3
14782
175.23
75239
17253820
0.25
Image 1
5592
55.62
51725
2679012
0.25
Image 2
6921
69.87
50267
3028912
0.25
Image 3
7590
74.23
47291
2561021
0
Image 1
12679
85.32
1775678
8012719
0
Image 2
13792
74.29
1745237
7800191
0
Image 3
13694
79.67
1865890
826102
Image J Values for All Samples
[Instructions: See worksheet page 8. To save time on typing a new Wiki table from scratch, use THIS TOOL to auto-generate a Wiki table: Excel-to-Wiki Converter. Copy the headers and values from the Excel spreadsheet you made, paste them into the form field, click submit, copy the Wiki code that the tool generated, and replace TABLE GOES HERE (below) with your auto-generated code.]
BME 100 Fall 2013
