SYBR Green Dye SAMANTHA
SYBR Green Dye is a membrane permeable nucleic acid dye used in molecular biology labs. The green dye can be placed in a sample with possible DNA and will glow a bright green if DNA is present. This is the most common use of the dye and helps qualitatively analyze if DNA is present in a substance. Although the DNA used in our samples was double stranded DNA, the SYBR Green Dye can also be used to detect single stranded DNA because it has such a high affinity. Although it was placed in the samples after being in the PCR machine, it also can be placed inside a PCR mixture and this allows the dye to bind to the single stranded DNA before it is synthesized by the Taq Polymerase. However, the result is the same. Either way it binds to DNA and glows when DNA is present.
Single-Drop Fluorimeter [A description of the single-drop fluorimeter device. Add a PHOTO for bonus points]
How the Fluorescence Technique Works SAMANTHA [In your own words, a summary of the information from page 9 of the worksheet]
Procedure
Smart Phone Camera Settings
iPhone 4s
Flash: Off Mode
ISO setting: N/A
White Balance: N/A
Exposure: N/A
Saturation: N/A
Contrast: N/A
Calibration
The distance between the smart phone cradle and the drop is approximately 10 cm.
Solutions Used for Calibration
Calf Thymus DNA solution concentration (micro/mL)
Volume of the 2X DNA solution (μL)
Volume of the SYBR GREEN I Dye solution (μL)
Final DNA concentration in 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
One must align the slide with the blue LED.
One should place 80 μL of SYBR GREEN in between two wells.
It is necessary for one to now add 80 μL of DNA sample in the drop that is formed from step #2.
One must cover this created sample from light in order to visualize the florescence.
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]
[insert: Shire_fluro.jpg] |100px]
Image J Values for All Samples
DNA Concentration
Area
Density
0
13856
225126
0
13856
51984
0
13856
68928
0.25
13856
199688
.025
13856
190251
.025
13856
273555
0.5
13856
405395
0.5
13856
482091
0.5
13856
636335
1
13856
1335751
1
13856
1274172
1
13856
1105412
2
13856
1895959
2
13856
1372946
2
13856
1769988
5
13856
1728032
5
13856
2042370
5
13856
1918408
Fitting a Straight Line [Place an IMAGE of your Excel plot with a line of best fit here. See worksheet page 8]