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.
Single-Drop Fluorimeter
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.
Single-Drop Fluorimeter
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.
Procedure
Smart Phone Camera Settings [Instructions: The type of smart phone you used and how you adjusted the camera settings, if applicable (see worksheet page 4).]
Type of Smartphone:
Flash:
ISO setting:
White Balance:
Exposure:
Saturation:
Contrast:
[Instructions: If you used an additional phone, describe the other type of smart phone you used and how you adjusted the camera settings, if applicable. If not, delete this part]
Type of Smartphone:
Flash:
ISO setting:
White Balance:
Exposure:
Saturation:
Contrast:
Calibration
[Instructions: Describe how to set up your camera in front of the fluorimeter. Add a PHOTO of this set-up for bonus points.]
Distance between the smart phone cradle and drop = 7 cm for Trial 1 , 4 cm for Trial 2
[Instructions: See worksheet page 6.]
Solutions Used for Calibration[Instructions: See worksheet page 6.]
row 1 cell 1
row 1 cell 2
row 1 cell 3
row 1 cell 4
row 2 cell 1
row 2 cell 2
row 2 cell 3
row 2 cell 4
row 3 cell 1
row 3 cell 2
row 3 cell 3
row 3 cell 4
[Add more rows as needed]
Placing Samples onto the Fluorimeter
[Instructions: Step one, in your OWN words]
[Instructions: Step two, in your own words]
[Instructions: Step three, in your own words]
[Instructions: Step etc., in your own words]
Data Analysis
Representative Images of Samples
[Instructions: Show an IMAGE where you drew a circle around the droplet with the freehand tool for a sample with no DNA]
[Instructions: Show an IMAGE where you drew a circle around the droplet with the freehand tool for a sample 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.]
TABLE GOES HERE
Fitting a Straight Line
[Instructions: Place an IMAGE of your Excel plot with a line of best fit here. See worksheet page 9]
BME 100 Fall 2013
