LAB 5 WRITE-UP
SYBR Green Dye
SBYR Green I dye is a cyanine dye used as a nucleic acid stain. When bound to DNA it absorbs blue light and emits green light. The stain binds to double stranded DNA (dsDNA) at very high levels and binds to single stranded DNA at much lower levels. This allows us to measure the amount of double stranded DNA while getting minimal noise on our signal from the presence of single stranded DNA. It can also stain RNA at lower levels, but this is not important for our experiment (in which we know our sample does not contain RNA). It is also the most sensitive stain available for detecting double stranded DNA during PCR.
This device is used to excite the stained DNA molecules in order to generate the designed signal, a green light, for us to capture with our camera. The single-drop fluorimeter is designed to hold a single drop of the sample and pass the wavelength ~497nm of light required to excite the stained DNA molecules within the sample.
How the Fluorescence Technique Works
A droplet is placed on the hydrophobic slide which allows the droplet to hold its spherical shape. The single droplet is then exposed to a blue light beam to excite the stained molecules which proceed to emit green light (our signal). We capture this signal (green light) with our smartphone camera. In theory, the amount of signal captured by our camera should be proportional to the concentration of DNA in the sample (the pictures must be filtered so that only the amount of green light captured is taken into consideration). Therefore, once we have calibrated our camera with known concentrations of DNA we should be able to compare signal strengths (green light emittance) from unknown DNA concentration samples with our calibration data to accurately estimate the DNA concentration in that sample.
Smart Phone Camera Settings
- Type of Smartphone: Samsung Galaxy SIII
- Flash: OFF
- ISO setting: 800
- White Balance: Auto
- Exposure: +2 (maximum)
- Saturation: Auto (no manual options)
- Contrast: Auto (no manual options)
The camera was placed upright in the supplied stand. The camera lens was positioned 6cm from the droplet in order to get the closest view possible while still being within the focus range of the camera. The height of the fluorimeter was adjusted so that the lens of the camera was at the same height as the droplet to ensure a full side view.
- Distance between the smart phone camera lens and drop = 6cm
Solutions Used for Calibration
| Calf Thymus DNA Solution Concentration (µg/mL) || Volume of the 2X DNA Solution (µL) || Volume of the SYBR Green I Dye Solution (µL) || Final DNA Concentration in 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
Placing Samples onto the Fluorimeter
- Place a clean slide with the hydrophobic side up into the slot in the Fluorimeter.
- Turn on the Fluorimeter.
- Use a micro-pipette to transfer 80 µL of SYBR Green I Dye and 80 µL of sample (either water or DNA solution) onto the slide to form a single droplet.
- Move the slide until the droplet is directly in line with the blue light beam (if not so already).
- Position camera at the predetermined position (see "Calibration") and set a countdown timer on the camera.
- Focus the camera on the droplet and start the countdown timer.
- Cover the entire apparatus with the supplied box to minimize external light and wait for the camera to capture the picture.
- Remove the box, open the recently captured picture and rename it to something that represents the sample DNA concentration and picture number.
- Remove the 160 µL droplet from the Fluorimeter slide with a micro-pipette and remove any remaining liquid with a paper towel.
- Repeat the above steps two more times to get a total of three pictures of three different droplets of the same DNA concentration.
- Repeat the above steps for each sample.
Representative Images of Samples
The picture below shows the control case where no DNA is present in the sample
The picture below shows a test case where DNA is present in the sample
Image J Values for All Samples
|CT DNA Final Concentration (µg/ml) || Area || Mean Pixel Value || RAWINTDEN Drop || RAWINTDEN Background || Corrected INTDEN
Fitting a Straight Line
- The below graphs illustrate the linear correlation between dsDNA concentration and fluorescence. The first graph shows the raw fluorescence data plotted against the known dsDNA concentrations, and the second graph shows the corrected fluorescence data (raw integrated density minus background integrated density) plotted against the known dsDNA concentrations.