LAB 5 WRITE-UP
SYBR Green Dye
SYBR Green Dye is used to verify the presence dsDNA in this lab. The dye fluoresces well with high concentrations of dsDNA,but fluorescence becomes less apparent as concentration decreases.
A single-drop fluorimeter is a device used to detect fluorescence.
Single-Drop Fluorimeter used in this lab
How the Fluorescence Technique Works
Some SYBR Green Dye is placed on a fluorimeter slide along with a sample of concentrated dsDNA and slid into the fluorimeter. The hydrophobic Teflon coating on the slide's rough surface allows the SYBR Green Dye and dsDNA to remain a droplet on the slide for easier measurement of fluorescence instead of being absorbed into the slide. A light short in wavelength and high in energy is shined on the sample. In this case, it would be the BlueLED. The blue light excites the molecules of the SYBR Green Dye in the presence of dsDNA to fluoresce and emit a bright green fluorescence glow. The lower the concentration of dsDNA, the lesser the excitation of the molecules and more dull the fluorescence.
Smart Phone Camera Settings
- Type of Smartphone: Android
- Flash: No flash
- ISO setting: 800
- White Balance: Auto
- Exposure: 2.0 (Highest)
- Saturation: No Saturation Setting
- Contrast: No Contrast Setting
First, the phone was placed on the cradle in front of the fluorimeter, about 8 centimeters away. Then plastic tops and other objects were used to adjust the height of the fluorimeter so that it lined up with the camera of the phone. A ruler was used to measure exactly 8 centimeters from the fluorescent light to the camera on the phone. After that, the phone was carefully focused on the droplet to provide a clear image for the analysis on ImageJ. Lastly, a black box was placed on top of the entire set-up, to block off any light.
Set-up of the lab
- Distance between the smart phone cradle and drop = 8cm
Solutions Used for Calibration
| 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
Placing Samples onto the Fluorimeter
- Place a slide, smooth side down, in the fluorimeter.
- Place 80µl of SYBR Green I solution in between the first and second row, in the middle circle.
- Make sure the use a new pipette tip every time to avoid contamination.
- Place 80µl of your sample solution on top of the SYBR Green I drop.
- Make sure to adjust the slide so that the blue fluorescent light is shining through the drop.
- Focus the camera on the phone and set the timer on the phone's camera.
- Close the cover of the box and get a picture of the fluorescence.
- Set the pipette to 160µl and remove the drop from the slide.
- Repeat the previous steps two more times for the same concentration.
- For every new concentration, use a new slide and repeat the same procedure.
Representative Images of Samples
The picture above is a sample with no DNA (no fluorescence).
The picture above is a sample with DNA. (fluorescence)
Image J Values for All Samples
|Calf Thymus DNA concentration (FINAL), microg/ml
||Mean Pixel Value
||RAWINTENDEN Of the Drop
||RAWINTDEN Of the Background
Fitting a Straight Line