LAB 2 WRITE-UP
SYBR Green Dye
SYBR Green Dye is a cyanine dye which binds to DNA. The result absorbs blue light and emits a green colored light. This stain works well mostly with double-stranded DNA, but it can still bind to single-stranded DNA. However, it will produce a green fluorescence less effectively with the single-stranded DNA.
A single-drop fluorimeter is a type of device that is used to measure patterns of fluorescence in relation to the number of molecules within a medium.
How the Fluorescence Technique Works
The fluorescence technique works by adding a fluorescent die to a liquid with a certain amount of DNA concentration. Teflon coated glass slides were used with a single-drop fluorimeter in order to produce spherical drops on the surface as a result of the hydrophobic properties created by the various circles of uncovered glass.
Smart Phone Camera Settings
- [iphone 4]
- Flash: Off
- ISO setting: Auto
- White Balance: Auto
- Exposure: Auto
- Saturation: Auto
- Contrast: Auto
The Camera was set up such that we could get the clearest image with maximum LED light absorption. A light box was placed over the fluorimeter to
darken the area. The Smart phone was aligned with the slide to get a sideways picture of the DNA sample. This creates the ideal angle to observe any
fluorescent light after the picture is taken. The camera phone is calibrated to view the fluorescent light with little interference from contrast and
recording the most light- done by setting the exposure high. The saturation is set to low so that color is not rendered in the photo. The ISO is set to high to maximize focus in the dark setting. With these settings, the fluorescence in the sample is easily observed.
- Distance between the smart phone cradle and drop = 6.5 cm
Solutions Used for Calibration
| Concentration Calf Thymus |
| Volume DNA added |
| Volume SABR GREEN I |
| Concentration resulting DNA|
| 5 || 80 || 80 || 2.5
| 2 || 80 || 80 || 1
| 1 || 80 || 80 || 0.5
| 0.5 || 80 || 80 || 0.25
| 0.25 || 80 ||80 || .125
| 0 || 80 || 80 || blank
Placing Samples onto the Fluorimeter
- 1.) With the Camera set in place we prepared the slide with SYBR Green I, a constant throughout the experiment
- 2.) 80 μL of 5μg/mL of calf thymus concentration DNA samples were then added to the slide
- 3.) To take 3 pictures in the the dark with as much of the blue LED light as possible, we used a timer on the camera, set to 3 seconds.
- 4.) The slide, now containing SYBER Green I and Calf Thymus DNA was then aligned in the cradle such that the light was shining into the DNA.
- 5.) With the Camera phone, we captured 3 images of the DNA and removed the light box.
- 6.) That sample was then disposed of with a micropipette
- 7.) Repeat with 80μL Green SABR I and 80μL of each Calf Thymus Concentration given by the first column in the table above
Representative Images of Samples
with DNA(positive signal)]
Image J Values for All Samples [See worksheet page 5]
| row 1 cell 1 || row 1 cell 2 || row 1 cell 3 || row 1 cell 4 || row 1 cell 5
| row 2 cell 1 || row 2 cell 2 || row 2 cell 3 || row 2 cell 4 || row 2 cell 5
[Add more rows as needed]
Fitting a Straight Line
| row 3 cell 1 || row 3 cell 2 || row 3 cell 3 || row 3 cell 4 || row 2 cell 5
[Place an IMAGE of your Excel plot with a line of best fit here. See worksheet page 8]