Name: Ashley Woodward
Name: Diljot Nagra
Name: Matthew Devera
Name: Fields Soumis Hyrkas
Name: Akihiko Ishihara
LAB 5 WRITE-UP
SYBR Green Dye
SYBR Green Dye is a small molecular dye that fluoresces well when put with dsDNA. However fluoresces very weakly in water or with single strands of DNA. This dye is used as a nucleic acid stain in molecular biology. When in the presence of DNA this dye binds with it and absorbs the blue light and emits green light.
A fluorimeter is a device that measures different amounts of fluorescence. Fluorimeters measure certain wavelengths and have two light detectors. One of these detects the absorbency while the other detects fluorescence emission.
How the Fluorescence Technique Works
This technique measures the concentration of DNA by adding a fluorescent dye and measuring the amount of fluorescence. We do this by using a single-drop fluorimeter. We put on a glass slide on the rough side and then put it in a light sensitive box. We set up the camera so it was looking at the slide horizontally and then set the timer for 3 seconds. Drops of SYBR Green are placed on the glass circles along with the DNA samples. Blue LED light from the fluorometer illuminates the drop this than causes the dye to fluoresce. The image that is taken is then analyzed to determine the concentration of DNA is in the solution.
Smart Phone Camera Settings
[Instructions: The type of smart phone you used and how you adjusted the camera settings, if applicable (see worksheet page 4).]
- Type of Smartphone: Galaxy S4
- Flash: Off
- ISO setting: Auto
- White Balance: Auto
- Exposure: 0
- Saturation: Auto
- Contrast: Auto
We set up the fluorimeter in the light sensitive box. We had a cradle for the phone in front of the device. We propped the phone so it was vertical. We added plates under the fluorimeter device until it reached the height of the camera level. As seen in the picture, the device was raised until the height was perpendicular with the camera on the phone.
- Distance between the smart phone cradle and drop = 5 cm
Solutions Used for Calibration [Instructions: See worksheet page 6.]
| Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL) || Volume of the 2X DNA colution (micoliters) || Volume of the SYBR Green 1 dye solution (microliters) || Final DNA concentration in SYBR Green 1 solution (micrograms/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 || 0
Placing Samples onto the Fluorimeter
- Place fluorimeter into shadow box to get a good picture of the fluorescent green
- Place slide into the fluormieter
- Set up phone so that the camera is looking straight onto the slide to get an accurate image
- Place the 80 microliters of DNA onto the slide
- Add the SYBR Green to the solution of DNA onto the slide
- Set a 3 second timer on your phone
- Close the flaps on the shadow box while pressing the camera button, make sure the box is completely shut
- Repeat for all concentrations of SYBR Green Dye until all concentrations are complete
- Take phone and upload all pictures online to separate the fluorescent green dye out on ImageJ.
Representative Images of Samples
Sample with no DNA
Image J Values for All Samples
Fitting a Straight Line
PCR Results Summary
Our positive control PCR result was 2.09 μL/mg.
Our negative control PCR result was -0.29 μL/mg.
Patient 28375: The final calculated PCR DNA concentration is -0.123 μL/mg. The sample appeared to have no visable sybre green in it.
Patient 23170: The final calculated PCR DNA concentration is 0.009 μL/mg. There was a trace amount of sybr green in the sample but not enough to qualify a positive test result.
The final conclusion is that patient 28375 is negative for the tested DNA sequence and patient 23170 is also negative based on the control value. The conclusions were made by comparing the patient DNA concentration values with the positive and negative control concentrations.