SYBR Green Dye
SYBR Green Dye is a small dye that is actively fluorescent in the presence of dsDNA but does not fluoresce very well with a single strand of DNA or in water. It is used primarily as a nucleic acid stain.
Single-Drop Fluorimeter
A fluorimeter (in this case one that uses the single drop technique) is a device that is used to measure and detect fluorescence by using a single drop of a specimen. It is a box shaped device that is meant to stay dark on the inside to use the blue light that is emitted to check for fluorescence.
How the Fluorescence Technique Works
The fluorescence Technique works by using optical caustics to measure the amount of fluorescence. Fluorescence happens when a molecule is excited and a longer wavelength is produced. Furthermore, the optical caustics causes the light to rise to the surface of the drop and the fluorescence to become more visible and making it easier to capture using a cell phone.
Procedure
Smart Phone Camera Settings
Type of Smartphone: iPhone
Flash: The flash was inactivated.
ISO setting: ISO was set to 800.
White Balance: White Balance was set to auto.
Exposure: Exposure was set to highest setting.
Saturation: Saturation was set to the highest setting.
Contrast: Contrast was set to lowest setting.
Type of Smartphone: iPhone
Flash: The flash was inactivated.
ISO setting: ISO was set to 800.
White Balance: White Balance was set to auto.
Exposure: Exposure was set to highest setting.
Saturation: Saturation was set to the highest setting.
Contrast: Contrast was set to lowest setting.
Calibration
1. Use the switch for the Blue LED to turn on the excitation light.
2. Check the settings on the smartphone if they can be adjusted and turn on the camera of your smart phone.
a. Turn the flash.
b. Adjust ISO to 800 or higher if possible.
c. Put white balance to auto.
d. Regulate exposure to highest setting.
e. adjust saturation to the highest setting.
f. adjust contrast to lowest setting.
3. To take a picture of the drop from sideways use the plastic trays to adjust the height of the fluorimeter after placing the smart phone on the cradle at a right angle from the slide. It can be helpful by filtering the blue light while using ImageJ.
4. In order to have smartphone cradle as close as the first two rows of the slide without having a blurry picture, one must adjust the distance between the smartphone cradle and the first-two rows of the slide. Keep the distance at least 4 cm away from the drop.
5. Using the ruler provided in the lab, record the distance between the smart phone cradle and drop. If there is a significant difference from one image to the next in the distances, the light collected will change slightly.
Distance between the smart phone cradle and drop = 7.2 cm
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
Using the plastic pipette, place a 80 microliter drop of SYBR GREEN I in the middle of the first two rows of the slide.
Add 80 microliter of one of the calf thymus or water blank solutions to the same place as drop of SYBR GREEN I.
Move the slide to place the drop so drop can be focused by blue LED light.
Take a picture after covering camera and fluorimeter by using the timer. Avoid stray light from the light box at the best possibility.
Focusing on drop take two pictures. Avoid moving the smartphone.
Take out the box without moving the smartphone.
To remove the 160 microliter drop from the surface use the pipette.
Move the slide to the next position.
Take three separate measurements of each drop of the same concentration of DNA in one of the slide positions and then move to the next concentration.
Several slides can be used if any mistake has made.
BME 100 Fall 2013
