SYBR Green Dye
SYBR Green dye was added to each of the samples from the PCR on the sample slides. A drop of each sample was placed onto a drop of this green dye each time the Fluorimeter was used. These drops of dyes react with different molecules of DNA. In the samples which had no DNA, these reactions didn’t take place. The reaction between the DNA and the green dye cause the blue excitation light to be absorbed and a green light to be emitted. Thus the samples with DNA shone green and the samples without DNA simply shone the original blue from the excitation light. The exterior black box, or light box, is used in order keep all other light from interfering with the images being captured.
The Fluorimeter allows for the quantitative evaluation of samples that underwent a Polymerase Chain Reaction (PCR). It is made up of an interior black box, or table, plastic trays, a slide port, a blue excitation light, a camera phone cradle, a Samsung galaxy s4 camera phone, and an exterior black box that covers all other components.
The first component, the interior black box, acts as both a table for the slide port and a house for the blue excitation light. The plastic trays sit under this interior box and allows researchers to correctly line up the slide samples and the camera they are using. The slide port is the narrow canal in which the sample slides sit. This port allows for the all samples to be easily interchanged. The blue excitation light is a light that is shone through the samples. The next component is the camera phone cradle and the camera phone (Samsung Galaxy s4). The camera phone is used to take high quality pictures of the samples so that they can then be transferred to a computer. The cradle simply holds the camera in one place. The last component is the exterior black box. This box covers all other components and keeps all unwanted light away from the experiment.
How the Fluorescence Technique Works
SYBR Green I is a DNA binding dye. In our lab experiment, it was added to samples after the DNA was amplified in a polymerase chain reaction (PCR). These samples were then placed in a Single-Drop Fluorimeter, where a laser/light was shown through the sample towards a camera. The dye attaches to the double stranded DNA and absorbs blue light while reflecting green light. In other words, it glows green when double stranded DNA is present, but it does not glow in water or in the presence of single stranded DNA. After doing a single drop fluorescence test, the amount of SYBR green dye, or green light emitted, was quantitatively graphed using a program called ImageJ. From these graphs, the concentration of DNA was calculated.
Smart Phone Camera Settings
Type of Smartphone: Samsung Galaxy S4
ISO setting: 800
White Balance: Auto
The camera was placed on the camera phone cradle. Plastic slides were placed under the interior black box (on which the sample slides sit), in order to line up the camera on the phone and the sample. The camera was placed 11.0 cm away from the drop in every sample in order to reduce error.
Distance between the smart phone cradle and drop = 11.0 cm
Solutions Used for Calibration
Initial Concentration of 2X Calf Thymus DNA Solution (μg/mL)
Final Concentration of 2X Calf Thymus DNA Solution (μg/mL)
Placing Samples onto the Fluorimeter
1. Place slide onto the slide port
2. Place 80 micro liters of the SBYR green dye onto the slide in the correct spot
3. Place 80 micro liters of the sample directly on top of the SBYR green dye
4. Shine the blue excitation light through the sample
5. Place the exterior black box, or the light box, over the other components
6. Take 3 pictures of each sample using the smart phone
7. Pipet the sample and dye off the slide and discard
8. Move the sample slide down so that the next area of the slide lines up with the camera
9. Using a new pipet tip for each sample, repeat steps 2-8 for each sample.
Representative Images of Samples
Image J Values for All Samples
PCR Product TUBE LABEL
INTDENS VALUES BASED ON 3 SEPARATE DROP MEASUREMENTS
Fitting a Straight Line [Instructions: Place an IMAGE of your Excel plot with a line of best fit here. See worksheet page 9]
PCR Results Summary
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 concentration of calf thymus DNA) to convert INTDEN values into DNA concentration. Your positive control and negative control samples are used to determine the threshold values for determining whether an unknown (patient) sample is truly positive or negative.
Positive control PCR result: 13.94 μg/mL
Negative control PCR result: 3.07 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 41731 F: All of this patient's samples illuminated to various degrees. The concentration values for this patient ranged from 3.21 to 5.04 μg/mL.
Patient 78042 M: None of this patient's samples illuminated. The concentration values for this patient ranged from 2.01 to 4.80 μg/mL.
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 41731 F: This patient's results were much closer to the negative control value, which indicates a negative result.
Patient 78042 M: This patient's results were much closer to the negative control value, which indicates a negative result.