LAB 5 WRITE-UP
SYBR Green Dye
SYBR Green Dye is a reagent used to stain DNA. After combining the SYBR Green dye with a double stranded DNA (Single stranded DNA works as well, but produces less effective results), the solution will be able to absorb blue light and emit green light. By measuring the green light, one will be able to measure how much DNA is present in the solution.
The single-drop fluorimeter device is a box that emits a blue light. A slot is present on the device for a glass slide to be placed on. Droplets of the SYBR Green dye and DNA solutions are placed on the glass slide for the light to be passed through.
How the Fluorescence Technique Works
The fluorescence technique works by combining a fluorescent dye to the DNA. In this lab, SYBR Green dye was used as the fluorescent dye. The droplets will remain in place due to the Teflon coated glass. A beam of blue light is emitted from the single-drop fluorimeter and is passed through the droplet. A fluorescent green light will be emitted from the droplet which will be measured by taking a picture on a smartphone and analyzing the picture file on ImageJ.
Smart Phone Camera Settings
Phone: Galaxy S3
- Flash: Off
- ISO setting: 800
- White Balance: Auto
- Exposure: Maximum
- Saturation: Maximum
- Contrast: Minimum
1. Adjust the camera settings on the smart phone so that they match the settings above.
2. Place smart phone in the cradle.
3. Make sure that the distance from the smart phone and the cradle is 11 cm.
Solutions Used for Calibration
|Calf Thymus DNA Concentration (Microg/ml)
||Volume of the 2X DNA Solution (ul)
||Volume of the SYBR GREEN I Assay (ng/mL)
||Final DNA Concentration in SYBR GREEN I Assay (ng/mL)
Placing Samples onto the Fluorimeter
- Before the sample can be placed on the Fluorimeter, the fluorimeter must be set up. This is done by first by placing the glass down on the fluorimeter; the glass must be placed so that the rough side is faced up. The fluorimeter must be turned on. Adjust the glass slide so that the blue light shines in the center of two rows. The hieght of the fluorimeter may need to be adjusted so that the camera has a clear view of the light.
- For the next steps, the micropipet will be needed. Begin by adjusting the volume on the micropipet to 80μL, and placing a new micropipet tip on the micropipet.
- Use the micropipet to gather 80μL of the SYBR Green I to be placed on the slide. The SYBR Green I will be placed on the glass slide for each sample. Be sure that the camera settings are all in place and take photograph of solely SYBR Green I, this is your blank.
- Next, use the micropipet to gather 80μL of the first sample, 0.25, and place it into the 80μL SYBR Green I present on the slide. Be sure that the light from the fluorimeter is in line with the droplet.
- Photograph the sample and SYBR Green I.
- Remove sample from slide with micropipet and repeat steps for each sample. After all samples have been collected (0,0.25,0.5,1,2,5) repeated this process two more times for a total of three pictures per sample.
Representative Images of Samples
Sample with no DNA
Sample with DNA
Image J Values for All Samples
|Calf Thymus DNA concentration (Final), Microg/ml
||Mean Pixel Value
||RAWINTDEN OF THE DROP
||RAWINTDEN OF THE BACKGROUND
Fitting a Straight Line