BME100 s2014:T Group10 L5
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OUR TEAMLAB 5 WRITE-UPBackground InformationSYBR Green Dye SYBR Green Dye is a dye that stains nucleic acids by binding to DNA or RNA. The dye is used to visualize DNA through staining agarose gels,
biochip applications, fluorescence imaging techniques, real-time PCR, and more. SYBR dye fluoresces when combined with dsDNA (Zipper Hubert Brunner 1). However, unbound SYBR Green is unreactive to light. This feature allows scientists to determine the concentration of a target DNA sample by measuring the intensity of the fluorescent light being emitted by the sample. The higher the intensity of the light being emitted, the more concentrated the sample is with the target DNA. SYBR Green, as its name implies, fluoresces with a green color. Accordingly, it is important to use a different wavelength of light to excite the sample in order to prevent distorting the results. There are several types of fluorescent dyes similar to the SYBR Green used in this lab, but SYBR Green was chosen for its environmental friendliness and high level of light emission intensity to allow for easier detection of binding to the target DNA.
A fluorimeter is a device used to measure and identify fluorescence in a medium. Fluorimeters measure certain wavelengths of light and have two light detectors, one of which detects absorbency while the other detects fluorescence emission (So Dong 1). Single-Drop Fluorimeters do this with a single drop of a sample that is placed on a hydrophobic slide in the path of a beam of single wavelength light. For example, in our setup we used blue LED light to allow for light absorption and prevent contaminated intensity data that would occur if we used a green LED since the SYBR Green Dye fluoresces in the green range. A light detector or camera placed at a uniform distance from this sample is then used to capture the intensity of the fluorescence being emitted from the sample. Cameras are acceptable for this laboratory set-up even though they are unable to measure light intensity because it is assumed that light intensity is proportional to brightness; the brighter an object is in a photo, the higher the light intensity.
How the Fluorescence Technique Works
Zipper, Hubert, Herwig Brunner, Jurgen Bernhagen, and Frank Vitzthum. "Investigations on DNA Intercalation and Surface Binding by SYBR Green I, Its Structure Determination and Methodological Implications." Ncbi.nih.gov. US National Library of Medicine, 12 July 2004. Web. 3 Apr. 2014. So, Peter TC, and Chen Y. Dong. "Fluorescence Spectrophotometry." Scribd. Macmillan Publishers Ltd, Nature Publishing Group, 16 Jan. 2009. Web. 03 Apr. 2014. ProcedureSmart Phone Camera Settings
[Instructions: Describe how to set up your camera in front of the fluorimeter. Add a PHOTO of this set-up for bonus points.]
[Instructions: See worksheet page 6.]
[Add more rows as needed]
Data AnalysisRepresentative Images of 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.]
Fitting a Straight Line [Instructions: Place an IMAGE of your Excel plot with a line of best fit here. See worksheet page 9]
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.
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