SYBR Green Dye
SYBR Green Dye is a detection probe that absorbs blue wavelengths of light and emits green wavelengths, enabling it to bind to double-stranded DNA. Although it performs well in the presence of double-stranded DNA, it fluoresces less effectively in water and with single-stranded DNA.
Single-Drop Fluorimeter
A Fluorimeter is a device used to quantitatively measure fluorescent material in a sample or substance in such a way that it is proportional to the amount of molecules being detected.
How the Fluorescence Technique Works
The components of a fluorimeter are designed specifically to enhance the environment of small, aqueous quantities for fluorescent analysis. The Teflon coated slides have multiple circles of bare glass that exhibit superhydrophobic properties which allow spherical drops to form on top of the glass. When light from the Blue LED in concentrated on the substance, the intensity excites the SYBR green Dye and causes it to fluoresce. As the dye combines with molecules such as dsDNA, it forms a structure that travels to the surface of the drop for observation. SYBR green fluorescence is a reliable technique because its sensitivity reacts favorably in its environment.
Procedure
Smart Phone Camera Settings
We used an iPhone 5
Flash:Off
ISO setting:NA
White Balance:NA
Exposure:NA
Saturation:NA
Contrast:NA
No additional phone used
A time delay setting was used, allowing 5 seconds for the light box to be closed before an image was captured.
Calibration
In order to measure unknown concentrations of DNA with the fluorimeter by measuring fluorescence, we must first measure the fluorescence of known concentrations of DNA using the same technique. To accomplish this, we used an assay of several known concentrations of calf Thymus DNA in conjunction with SYBR green dye. We took pictures of the fluorescing drops and used imaging software to measure the fluorescence. SYBR green dye fluoresces green and the ImageJ software allows the colors of one picture to be split into pictures of the component colors. We can then select just the green light and measure the pixel density, allowing us to quantify the fluorescence. With a known concentration and a known pixel density, we can then define a relationship between the two which will allow us to later measure the fluorescence of an unknown sample to determine the DNA concentration of it.
We inserted the iPhone inside a cradle that gave us a right angle view. Afterward we adjusted the height of the phone so that it is as even with the drop on the slide.
The image above shows the set-up used. The camera phone in the fore ground is focused on the large drop of dye and sample on the fluorimeter behind it. The drop is lit by the blue LED light and the rest of the fluorimeter is darkened by the surrounding light box
Distance between the smart phone cradle and drop = 3cm
Solutions Used for Calibration
Calf Thymus DNA solution (microg/mL)
Volume of DNA Solution (μL)
Volume of SYBR GREEN I Dye solution (μL)
Final DNA concentration in PicoGreen Assay (ng/mL)
0
80
80
blank
.25
80
80
.125
.5
80
80
.25
1
80
80
.5
2
80
80
1
5
80
80
2.5
Placing Samples onto the Fluorimeter
Align light between first two clean rows of spots so it will shine directly at the drop, secure slide with tightening screw
Using a clean micro-pipette tip, slowly place 80μL of SYBR green dye allowing it to span between both spots
Using a clean micro-pipette tip, add 80μL of the sample solution to the SYBR dye
Complete assembly by placing the camera/cradle in position and cover with light box
Start timed delay picture on phone camera
Close light box quickly and carefully, then wait the allotted time for the picture to complete
Extract and discard solution from the slide
These steps were repeated in triplicate for each of the samples listed above.
BME 103 Spring 2013
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