LAB 5 WRITE-UP
SYBR Green Dye
SYBR Green Dye is a high-sensitivity, double-stranded DNA binding reagent dye that is used as a nucleic acid stain in molecular biology. It is used for detecting double-stranded DNA generated during PCR. These stains provide sensitivity with low background fluorescence, and because the stain binds to double-stranded DNA, the DNA-dye-complex absorbs blue light and emits green light. Because of this characteristic, we are able to detect how much DNA is present in a solution.
The single-drop fluorimeter device is a box that emits a blue light. A slot is present on the device for a sample slide to be placed on. The surface of the sample slide on which the solution can be dropped on is hydrophobic, and because of this water (when dropped on the surface) forms a sphere which can be observed for testing the presence of DNA. Then the SYBR Green dye and DNA solutions are placed on the sample slide, and blue light passes through the solution so that green light will be emitted. This process enables users to measure DNA in fluorescent materials.
How the Fluorescence Technique Works
In this experiment, the fluorescence technique enables students to view a controlled environment for fluorescent analysis. This environment is enclosed in darkness, which allows for easier detection of DNA in solutions. The sample slides on which solutions are placed on are hydrophobic, which means that when liquid samples are dropped on the surface they form spheres. A blue light is concentrated onto the substance and this causes the SYBR Green Dye to emit a green light. This process forms a structure which travels to the surface of the sample so that it can be observed in the experiment.
Smart Phone Camera Settings
- Type of Smartphone: Samsung Galaxy S4
- Flash: Off
- ISO setting: Auto
- White Balance:Auto
In this lab, a fluorimeter was required to detect fluorescence. This instrument measures how the quantity is related to the amount of florescent material and, less directly, proportional to the amount of the molecule being detected. First, find the smooth side of the glass using gloves. Next, turn on the fluorimeter and place the slide into the fluorimeter with the smooth side down. Set the camera timer to 3 seconds and place the phone on the cradle. Adjust the height of the fluorimeter so that the camera view of the slide nearly edge on. Place a 80 uL drop of SYBR Green I solution in the middle of the first 2 circles on the slide. Then place the same amount of the Sample Calibration solution on top of the SYBR Green solution. Adjust the slide so that the light illuminates the center of the drop. Make sure to adjust the distance between the smartphone and fluorimeter to greater than 4cm away and record the distance. Keeping the drop in focus, cover with the light box keeping one flap open. Initiate the timer and close the flap before the picture is taken. Remove the drop from the slide using the micro pipette and then repeat the same procedure with the next two clear circles for each sample calibration solution.
- Distance between the smart phone cradle and drop = 7 cm for Trial 1 , 4 cm for Trial 2
Solutions Used for Calibration
| Calf Thymus DNA solution concentration (microg/mL) || Volume of the 2X DNA Solution (μL) || Volume of 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"
- Step one: First place a 80 uL drop of SYBR Green I solution in the middle of the first 2 circles on the slide.
- Step two: Add 80 uL of the sample calibration solution on top of the drop.
- Step three: Make sure the light illuminates the center for the drop in the middle of the 2 clear circles.
- Step four: After the picture is taken, remove the drop with a micro pipette and throw the waste tip into the red cup to be disposed of in the bio-hazard can. Repeat procedure a total of three times for each concentration of calibration solution.
Representative Images of Samples
Sample Image with no DNA
Sample Image with DNA (positive signal)
Image J Values for All Samples
[Instructions: See worksheet page 8. To save time on typing a new Wiki table from scratch, use THIS TOOL to auto-generate a Wiki table: Excel-to-Wiki Converter. Copy the headers and values from the Excel spreadsheet you made, paste them into the form field, click submit, copy the Wiki code that the tool generated, and replace TABLE GOES HERE (below) with your auto-generated code.]
|Calf Thymus DNA concentration (FINAL), microg/ml
||Mean Pixel Value
||RAWINTDEN OF THE DROP
||RAWINTDEN OF THE BACKGROUND
| 0 || 100416 || 17.7 || 1777348 || 78918
Fitting a Straight Line
[Instructions: Place an IMAGE of your Excel plot with a line of best fit here. See worksheet page 9]