LAB 5 WRITE-UP
SYBR Green Dye
SYBR dye is a dye that glows strongly in the presence of double stranded DNA and grows weaker in the presence of single stranded DNA. This dye is used to stain the DNA produced during polymerase chain reaction and consequently can be used analyze how accurately and efficiently the PCR runs The DNA dye is able to absorb the blue light being given off by the light and turns green when a double stranded DNA has been detected. The SYBR dye stains the dsDNA that has been made through the process of polymerase chain reaction (PCR).
Single-Drop Fluorimeters are used to measure fluorescence by analyzing the type and the amount of fluorescent molecules in a substance. For example, if SYBR is mixed with DNA, the DNA will enable the SYBR to fluoresce and consequently the device will be able to detect the amount of DNA retrieved from PCR. Below is an image of the fluorimeter device.
How the Fluorescence Technique Works
A drop of the dye is placed on the hydrophobic slide in the fluorimeter device. The hydrophobic nature of the slide causes the dye to clump together in one small droplet. When the DNA is added to the dye, the dye will enable the DNA to fluoresce. As the concentration of the DNA increases, so does the amount of green light emitted. Thus, when they are mixed together, the amount of DNA and fluorescence will have a directly proportional relationship. When the drop of the mixture of SYBR/DNA is placed on the slide, the light beams from the side make it easier to see the fluorescence by nullifying the noise from the environment. Then, the fluorescent substance that is placed on the slide generates light of a short wavelength. Then, the energy is released in the form of photons. The amount released will determine the concentration of the samples and will be indicated by the amount of fluorescence in the pictures.
Smart Phone Camera Settings
- Type of Smartphone: iPhone 5
- Flash: Off
- ISO setting: Highest (Number not displayed on app)
- White Balance: 5000 K (Neutral)
- Exposure: High
- Saturation: High
- Contrast: Low
The image below shows the distance between the phone cradle and the drop.
- Distance between the smart phone cradle and drop = 9 cm
The image below shows the complete set up with the black box. A picture of a sample was about to be taken
Solutions Used for Calibration
|Calf Thymus DNA Solution Concentration (microg/mL)
||Volume of the 2X DNA Solution (μL)
||Volume of SYBR GREEN I Dye Solution (μL)
||Final DNA Concentration in SYBR GREEN I Assay (ng/mL)
Placing Samples onto the Fluorimeter
- The slide was slid into the fluorimeter with the rough side face-up
- The fluorimeter was tuned on and the slide was adjusted so the blue light fell between the two rows of dots
- The micropipette was set to 80 microliters
- A clean tip was attached to the micropipette
- The micropipette was used to transfer the SYBR Green microtube to the slide between the first two rows of dots
- The pipette tip was thrown into the waste basket and a clean tip attached in order to transfer calibration solution onto the slide.
- The calibration solution was placed on top of the slide on top of the SYBR Green.
- A picture of the fluorimeter was taken
- The micropipette was used to remove the drop of SYBR and calibration solution
- The micropipette tip was thrown in the waste basket
- This process was repeated 5 times to confirm that all concentrations of calibration solution are calibrated
- Every time it was repeated, a different location on the slide was used
Representative Images of Samples
This image represents the area of the droplet with 0 DNA being analyzed for green color intensity.
This image represents the area of the droplet with a 2.5 M final concentration of DNA solution being analyzed for green color intensity.
Image J Values for All Samples
|Calf Thymus DNA concentration (FINAL) micrograms/mL
||Mean Pixel Value
||RawIntDen of the Drop
||RawIntDen of the Background
Fitting a Straight Line
This image shows that the intensity of the SYBR dye increases as the concentration of DNA increases. The only concentration that was unusual in the experiment was the 0.50 concentration of DNA. The rest of the values on the graph increased accordingly.