BME100 f2013:W900 Group7 L5
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LAB 5 WRITE-UP
SYBR Green Dye
The SYBR Green Dye is a small molecular dye that fluoresces, or glows, very well in the presence of dsDNA (double stranded DNA). In this experiment, SYBR Green I was used, which has maximum efficiency/transmittance at a wavelength of approximately 500 nm, which lies in the range for green light. Therefore, when the SYBR Green Dye binds to the dsDNA and is excited, it strongly emits green light. The quantification and measurement of this green light was performed by taking pictures with a smartphone from a constant distance away from the single-drop fluorimeter.
The single-drop fluorimeter was used to detect the amount of fluorescence in each sample. It is a small, box-shaped device with a thin, long slot. A slide with a rough, superhydrophobic surface and circles of glass was placed in this slot. The device also emits a blue light--the purpose and role of this blue light in the fluorescence technique is explained in the next section.
How the Fluorescence Technique Works
The fluorescence technique is based on the properties of the slides placed in the single-drop fluorimeter device. These slides have a rough surface made of Teflon that is superhydrophobic as well as small circles made of glass. Because DNA is polar, and hence hydrophilic, it is repelled by the superhydrophobic surface and binds to the small glass circles on the slide due to their adhesive properties. Therefore, adding DNA to the slide causes the DNA to form small spherical globules, rather than spread all over the slide. This allows two things to happen: first, light from the Blue LED of the single-fluorimeter device is more strongly focused onto the DNA, thereby exciting the SYBR green dye and increasing the fluorescence event; two, the solvent added along with SYBR Green I forms a complex with DNA and prefers to go to the surface of the globule.
Smart Phone Camera Settings
Place the smartphone in the cradle at a right angle from the slide. Adjust the height of the fluorimeter using the plastic trays to achieve the 90° angle. Adjust the distance of the cradle to the fluorimeter so that the phone is as close to the slide as possible so you get a clear picture (minimum of 4 cm). Be careful not to shift the phone and fluorimeter setup too much from its fixed state during the experiment, as it can affect the difference between each pictures of globule samples.
Placing Samples onto the Fluorimeter
Representative Images of Samples
This image is of a sample containing no DNA.
This image is of a sample containing a 2.5 microg/mL solution of DNA.
Fitting a Straight Line