SYBR Green Dye SYBR Green dye was the indicator that was utilized in the experiment in order to detect particular strands of DNA. When added to a substance, we can determine the presence of double-stranded DNA. This type of florescent dye does not necessarily turn the substance green, instead it emits a green light when the appropriate wavelength of light shines on it. The reasoning behind this phenomenon is because the florescent dye strongly binds to double stranded DNA, making it easier to visualize it and detect it. Although it can easily detect the presence of double-stranded DNA, the SYBR Green Dye fluoresces very weakly or may not even fluoresce at all in substances such as water, or in the presence of single-stranded DNA.
Single-Drop Fluorimeter The Single-Drop fluorometer was the instrument that was utilized in the process of our experiment. This device is generally used to measure the parameters of fluorescence. The device is composed of a portable light box which is used to shine light through the SYBR Green solution, and a black portable box that makes it easy to capture pictures in the dark. In this experiment, our personalized fluoromoter was used to detect the emission of SYBR Green when added to a specific substance. If double-stranded DNA was detected, the light shining through the substance would turn green.
How the Fluorescence Technique Works In the experiment, we used a single-drop fluorometer to detect the presence of double stranded DNA. This process is composed of a small slide that has a superhydrophobic surface (surface that repels water), and a light box used to visualize the type of light reflected from the drop. When high concentrations of DNA were added to the SYBR Green solution, all wavelengths of light were absorbed except green. However when single-stranded DNA or water was added, the blue LED-light did not illuminate any particular color.
Smart Phone Camera Settings
Type of Smartphone: Apple iPhone 5S
ISO setting: Auto
White Balance: Auto
The camera was set up by placing the iPhone 5S in the holding compartment of the lab equipment. It was measured to be 8 centimeters away from the origin of the light emission. The settings (stated above) were adjusted; the height of the fluorimeter was adjusted in order to achieve a perfect side-angle perspective of the "beach ball" shaped liquid drop.
Distance between the smart phone cradle and drop = 8 centimeters
Solutions Used for Calibration
Initial Concentration of 2X Calf Thymus DNA solution (μg/mL)
Volume of the 2X DNA solution (μL)
Volume of the SYBR GREEN I
Final DNA concentration in SYBR Green I solution (μg/mL)
Placing Samples onto the Fluorimeter
Before placing samples onto the fluorimeter, turn on the Blue LED light & have the camera on the smartphone ready.
Insert the glass sampling surface, with the smooth side facing down.
Position the glass sampling surface so that the light emits between the first two rows of the glass.
Place an 80 μL drop of SYBR Green I in the middle of the first two rows.
Swap out the pipette tip with a new tip.
Place an 80 μL drop of a specifically concentrated Calf Thymus DNA solution onto the top of the previously placed SYBR Green I drop. Every three 160 μL sample drops will vary in concentrated Calf Thymus DNA solution.
Place the black screen over the camera, set the camera to take a picture (on a timer) after the lid is shut, take the picture, and proceed towards the next sample drop.
In order to place a new sample drop, remove the 160 μL solution, move the glass sampling surface to the next two available rows, and repeat the process of placing two new 80 μL drops onto each other (as stated before).
Representative Images of Samples
Sample w/o DNA
Sample w/ DNA
Image J Values for All Samples
Final DNA Concentration in SYBR Green I Solution (μg/mL)
Mean Pixel Value
RAWINTDEN of the Drop
RAWINTDEN of the Background
RAWINTDEN Drop - RAWINTDEN Background
Fitting a Straight Line
PCR Results Summary
Volume of the DILUTED PCR Product solution (µL)
Volume of the SYBR GREEN I
INTDENS VALUES BASED ON 3 SEPARATE DROP MEASUREMENTS
AVERAGE INTDENS VALUE
Dye solution (µL)
PCR Product Tuble Label
Average INTDENS Value
PCR Product Concentration (μg/mL)
Corrected PCR Product Concentration (μg/mL)
Instructor's summary: You completed 8 PCR reactions in a previous lab. You used the SYBR Green I staining and imaging technique to measure the amount of amplified DNA in each PCR reaction. You used a standard curve (based on known concentrations of calf thymus DNA) to convert INTDEN values into DNA concentration. Your positive control and negative control samples should be used as threshold values for determining whether an unknown (patient) sample is truly positive or negative.
Your positive control PCR result was 0.877657887 μg/mL
Your negative control PCR result was -0.04803684 μg/mL
Write-in each patient ID and give both a qualitative (what the images looked like) and a quantitative description (μg/mL) of what you observed.
Patient 26734: average value of 0.261416499 μg/mL
The images of the sample drops had little accents of green that were present.
Patient 22707: average value of 0.3457359472 μg/mL
The images of the sample drops had a small hint of green, yet still greater than Patient 26734.
Compare each patient's results to the positive control value and the negative control value. Draw a final conclusion for each patient (positive or negative) and explain why you made that conclusion.
Patient 26734: The results concluded that the test was inconclusive because Patient 26734 produced results that had a substantial amount of PCR Product Concentration; however, not great enough to conclude that the patient contains the sought-out after DNA strands.
Patient 22707: The results concluded that the test was inconclusive because Patient 22707 produced results that had a greater PCR Product Concentration than Patient 26734; however, not great enough to conclude that the patient contains the sought-out after DNA strands.
Each patient was found within the middle of the range between the positive control (0.877657887 μg/mL) and the negative control (-0.04803684 μg/mL); thus, further tests would need to be performed in order to reach a conclusion on whether or not these two patients may or may not have the DNA strands that the lab investigation was intended towards looking for.