LAB 5 WRITE-UP
SYBR Green Dye
- SYBR Green dye is a small molecular dye that fluoresces very well in the presence of dsDNA (double strain DNA) but fluoresces very weakly in water or with single stands of DNA.
- SYBR Green dye works on the principal of molecular bonding, in the way that it bonds with double bonded strains of DNA more that the rest of the weakly copied single strands.
- There were 6 different DNA's that we have to mix with the Green SYBR, then observe the change of color while mixing the Green SYBR with each DNA separately.
Single Drop Fluorimeter
- A Fluorimeter is an instrument that detects fluorescence (a very sensitive detection technique that uses the generation of light by a molecule due to excitation by shining light of a shorter wavelength) quantity
- 6 drop trails were conducted for each sample. The Green Syper with different DNA sample ,each time. The change of color was obvious. The color was gradually changing to green as you can see from these images.
How The Fluoresces Technique Works
- The Fluoresces Technique works in various steps. First step that is taken in this process, is the action of the SYBR Green Dye infiltrating the dsDNA and binding to the center of the acid by molecular means. After this is completed the Green dye just sits and waits until it has been excited by an outside force. This force is fulfilled in the way of the blue light emitted from the Single Drop Fluorimeter. The SYBR Green Dye has a lower frequency than the blue light emitted, so the blue acts upon the Green Dye rather than the other way around. This is in turn recorded by a camera phone and analyzed by the ImageJ software. This software provides the most accurate information about light and it's gradients.
- The Fluorescence Technique Worked perfictly. The color was changing to green gradually everytime we mix one of the DNA's with the Green SYBR.
Smart Phone Camera Settings
- Type of Smartphone: Samsung Galaxy series 4
- ISO setting:auto
- White Balance: auto
The camera phone was set up several centimeters away from the fluorimeter, by placing it into a cradle. One of the first problems was that the galaxy S4 was very large, therefore the fluorimeter need to be placed at a much higher level just to look directly into the camera lens.
- Distance between the smart phone cradle and drop = 4.0 cm
[Instructions: See worksheet page 6.]
Solutions Used for Calibration [Instructions: See worksheet page 6.]
| Calf thymus DNA solution concentration (microg/mL) || Volume of the 2x DNA solution (uL) || volume of the SYBR GREEN i Dye solution (uL) || final DNA concentrationin 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
[Add more rows as needed]
Placing Samples onto the Fluorimeter
- using a micropipette measure 80 um of SYBR GREEN and please this on to 2 of the clear dot in the middle row of the slide, making sure to wipe off that area after each trial.
- using a new tip on the micropipette please 80 um SAMPLE/CALIBRATION on top of the droplet of SYBR GREEN
- carefully move the slide until the blue light hits the middle of the drop and reflects the light crossed to the opposite side
- move the smartphone around to achieve focus without moving the phone more than 4 cm from the slide (Record this distance)
- carefully placed the light box over this set up cheap one flap of the box
- check the focus of your smartphone one more time
- set a timer for 5 seconds on your smartphone to take a picture and close the flap on the box before it takes the picture
- using the pipette dispose of the 160 um droplet in the liquid waste container
- take the paper towel and clean off the area used.
- move the slide to center in between the next two clear dots
- repeat this process two more times for this concentration using different pairs of cleared dots on this slide
- go through this entire process three times for each concentration needed using clean slides for each new concentration
Representative Images of Samples
Fitting a Straight Line
[Instructions: Place an IMAGE of your Excel plot with a line of best fit here. See worksheet page 9]