LAB 5 WRITE-UP
SYBR Green Dye
The SYBR Green Dye, is a stain that fluoresces and binds in the presence of double stranded DNA, but does not bind or fluoresce in water or to single strand DNA. The DNA dye absorbs blue light and emits green light when bound to double stranded DNA. This method is used to measure PCR products. Since the PCR produces millions of copies of DNA, the SYBR Green Dye then binds to each after the process is over resulting in a net increase of fluorescence.
A Single-Drop Fluorimeter device detects fluorescence. The device detects the the visible light emitted, measures fixed wavelengths and measures patterns of florescence in relation to the amount molecules.
How the Fluorescence Technique Works
The main parts of the fluorescence device is the slide and the blue LED light. The slide is covered with Teflon which is used mainly for its superhydrophobic properties meaning it has extremely low coefficient of friction. There are also little circles of bare glass all over the slide. This becomes helpful when placing the drops of your sample because the drops cling to the glass circles but at the same time are repelled by the hydrophobic surface. This structure creates a perfect environment for two things to happen. The first is that when the drops are placed, there is a Blue LED light from one side that is concentrated on the sample causing an increase in intensity which in turn increases the fluorescence. The second is that by using SYBR Green 1 dye mixed with the sample, the dye binds to the double stranded DNA making it go to the surface. We can then see the fluorescence when we take a picture of the sample in the dark.
A picture of our Fluorimeter device
Smart Phone Camera Settings
- Type of Smartphone: iPhone
- Flash: Off
- ISO setting: 800
- White Balance: Auto
- Exposure: Highest
- Saturation: Highest
- Contrast: Lowest
The smart phone was placed on the cradle at a right angle from the slide and the height of the fluorimeter was adjusted using the plastic trays so that the camera took a picture of the drop sideways.
- Distance between the smart phone cradle and drop = 4 cm
A picture of our camera setup:
Solutions Used for Calibration
Placing Samples onto the Fluorimeter
1. Take a clean slide, with the superhydrophobic side up, and insert it into the single drop fluorimeter.
2. Take a clean tip and use it to get 80 uL of SYBR green solution which will be put onto the two clean middle circles closest to the front.
3. Take a new clean tip and place 80 uL of the sample DNA on top of the SYBR Green solution.
4. Adjust the slide so that the blue light on the fluorimeter is passing through the middle of the drop.
5. Once the slide is in position, place it into the box and make sure it is dark.
6. Place an 80 microliter drop of SYBR green I in the middle of the first two rows of the slide using the plastic pipette. Then add 80 microliters of one of the calf thymus solutions.
7. Align the drop by moving the slide so that the blue LED light is focused by the drop to the middle of the black fiber optic fitting on the other side of the drop.
8. After covering the fluorimeter and camera with the light box, use the timer on the smartphone to take a delayed picture of the drop.
9. Take two images of the drop were and be sure the smartphone was not moved to insure consistency.
10. Open the box and dispose of the sample using the pipette.
11. Take three separate measurements of a “sample” of the same concentration of DNA in one of the slide positions, move to the next concentration and repeat.
Representative Images of Samples
The image above shows the circle drawn around the droplet containing no DNA.
The image above shows the circle drawn around a sample containing DNA.
Average Image J Values for All Samples
| DNA Concentration (μg/mL)
Fitting a Straight Line