SYBR Green Dye
SYBR Dye is a photosensitive molecular dye that exhibits a green fluorescence in the dark when added to enough single-stranded DNA.
A Single Drop Fluorimeter is a device that shines blue light through a drop sample that is placed on a glass slide and detects fluorescence.
How the Fluorescence Technique Works Since using the naked eye to view if a sample has DNA is near to impossible; a fluorescent dye is used.
A camera makes the florescent dye even easier to see, and creates images which can be analyzed for amount of dye present.
The way the fluorescence works is by utilizing a green dye that activates only under the presence of double stranded DNA (or dsDNA).
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: Samsung Galaxy S2
ISO setting: Default
White Balance: Auto
The camera phone was set up in the cradle with an angle to view the side of the drop. The fluorimeter was raised about 1 1/4 inches to accomodate for the phone height.
Distance between the smart phone cradle and drop = 6.5 cm
The phone was removed from this image to simplify and completely show distance setup.
Solutions Used for Calibration
Distance of Phone from Fluorimeter
Initial Concentration of 2X Calf Thymus DNA solution (μg/mL)
Volume of the 2X DNA solution (μL)
Volume of the SYBR Green I Dye Solution
Final DNA concentration in SYBR Green I Solution (μg/mL)
Placing Samples onto the Fluorimeter:
1. Turn on the flourimeter. Place the super hydrophobic slide on the flourimeter.
On the hydrophobic side of the flourimeter, place a 80 microliter drop of SYBR GREEN I on the middle rows of the slide.
2. Now on the sphere of dye that is on the slide, add 80 microliter of one of the solutions being tested. This will now react or not.
3. Next the flourimeter will shine a blue light, align the slide so that the drop sample is in the middle of this light.
4. Acquire a box that can cover the flourimeter in order to create a dark environment.
Place a smartphone in the cradle, and use a timer on the camera to take a picture of the drop. Once the timer starts quickly put the box over the whole setup, so that outside light does not interfere.
Representative Images of Samples
High DNA - using green channel
Low DNA - using green channel
High DNA Concentration
Low DNA COncentration
Image J Values for All Samples
Final DNA Concentration
Mean Pixel Value
Rawintden of the Drop
Rawintden of the Background
RAWINTEDEN (drop) - RAWINTDEN (background)
PCR Results with Corrected COncentration Values
Average INTDENS Value
PCR Product Concentration (µL/mL)
Corrected PCR Product Concentration (µL/mL)
Fitting a Straight Line
Intends values for each Calf Thymus DNA cocnentration.
PCR Results Summary
A Polymerase Chain Reaction (PCR) was completed for 8 DNA samples--3 DNA samples from each of two patients and a positive and a negative control sample. After the reactions proceeded and cooled down, the samples were tested on a fluorimeter for amplification of DNA using SYBR Green I as a stain. The controls were necessary to ensure amplification results for the patients' samples had a comparative standard and the results were due to the procedure and not an external affect. Using a known concentration of DNA from the calibration phase, a standard curve was created that was a basis for conversion from imageJ INTEN values to DNA concentrations for the PCR samples.
Your positive control PCR result was 90.744 μg/mL
Your negative control PCR result was -2.770 μg/mL
Patient 1, ID: 51917 samples hardly showed any visibly show green
Patient 2, ID: 17539 all of their samples showed little to no green dye.
Patient 1, ID: 51917 samples were negative, were a bit blue but had little to no green, and would therefore also be considered negative for the diseased DNA. The average DNA concentration for Patient 1 was -1.824 μg/mL.
Patient 2, ID: 17539 samples were mostly negative, with an average concentration of -2.077, but one of the samples showed a bit of green with a calculated DNA concentration much farther from the negative control than the other results, but still farther from the positive control. The test would be considered negative for the diseased DNA, but with the one sample in disagreement with the other two, a retest may be desired to ensure that the patient is negative for the diseased DNA.