SYBR Green Dye
SYBR Green Dye is used as a nucleic acid stain when performing tests that involves DNA. The stain binds to double stranded or single stranded DNA, but works best when binded to double stranded DNA. The SYBR dye is used to detect DNA that results from a PCR test. Since the stain binds to DNA, the resulting solution will absorb blue light and emits green light. Depending on DNA concentration, the vibrance of the color will vary.
Single-Drop Fluorimeter
A single-drop fluorimeter device is a box that has a specific section that emits blue light. In order to center the slide in the middle of the beam of light, there is a slot that holds the slide in place, but allows it to be adjusted when necessary. The slide that is used has a hydrophobic surface, which keeps the solution in a spherical shape rather than spreading across the whole slide. After the SYBR Green Dye is added to the surface of the slide, the DNA sample is added on top of the dye. The resulting solution, in a perfect sphere shape, can then be centered in the beam of light by adjusting the slide. When the blue light passes through the solution, green light is emitted, due to the dye present in the soluion.
How the Fluorescence Technique Works
The fluorescene technique works in this experiment because of multiple factors. The fluorimeter (and the solution on the slide) is completely enclosed which creates a completely dark environment, allowing for easier detection of DNA in any particular solution. The solution on top of the slide will form a sphere (due to the slide's hydrophobic nature), which allows the solution to stick to itself rather than spreading across the whole slide. A beam of blue light is then concentrated on the middle of the solution sphere, which causes the solution to emit green light, due to the presence of SYBR Green Dye. The different amounts of emitted light can then be observed and recorded, which correspond to different concentrations of DNA that are found on the slide.
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 4s
Flash: Flash OFF
ISO setting: n/a
White Balance: n/a
Exposure:n/a
Saturation: n/a
Contrast: n/a
[Instructions: If you used an additional phone, describe the other type of smart phone you used and how you adjusted the camera settings, if applicable. If not, delete this part]
Type of Smartphone: Iphone 4s
Flash: Flash OFF
ISO setting: n/a
White Balance: n/a
Exposure: n/a
Saturation: n/a
Contrast: n/a
Calibration
[Instructions: Describe how to set up your camera in front of the fluorimeter. Add a PHOTO of this set-up for bonus points.] The phone is placed into a holder that will secure it straight up and down to view the slide from the perfect angle. The Flourimeter and phone are both adjusted for the optimal picture height and angle. After the angle is set, an enclosed box is set over the whole system to keep out light and make the perfect conditions for a photo of the solution. The system must be completely dark when the picture is taken so that we are able to see the light flow through the DNA and SYBR green solution.
Distance between the smart phone cradle and drop =
Both of the Iphone 4s's were set at 5.5 cm away from the solution drop.
Solutions Used for Calibration[Instructions: See worksheet page 6.]
Concentration of Calf Thymus DNA solution (microg/mL)
Volume of the 2x DNA Solution (μL)
Volume of the SYBR Green Dye Solution (μL)
Final DNA Concentration in SYBR Green 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
0
[Add more rows as needed]
Placing Samples onto the Fluorimeter
Step 1: Take the multi celled wall slide and place it on the Single-Drop Flourimeter so that the light being emitted is able to pass through two of the rows on the multi celled wall slide.
Step 2: Take the micropipette and draw out 80 microliters of the SYBR green solution, and then slowly release the solution onto the two rows of the slide so that the light is able to penetrate through the solution. Then dispose of the tip of the micropipette into a waste disposal container.
Step 3: Place a new tip onto the end of the micropipette and draw 80 microliters of the Calf Thymus solution.
Step 4: Take the micropipette and carefully release the Calf Thymus solution on top of the SYBR green drop that is on the slide. Be careful not to let any solution spill over and make a second drop on the slide. Dispose of the tip on the micropipette into a waste container.
Data Analysis
Representative Images of Samples
No DNA
With DNA
Image J Values for All Samples
Fitting a Straight Line
As the concentration of the SYBR Green Dye increase so does the vibrance of the color emitted.The Intden of the drop represents the integral of the density. The graph has a strong linear fir the first set of points. The last three point were a slightly off but still matches the graph and worksheet well.
BME 100 Fall 2013
