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.
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.
[[Image:Photo(1).JPG]]
[[Image:Photo(1).JPG|Test Caption]]
* Distance between the smart phone cradle and drop = 3cm
* Distance between the smart phone cradle and drop = 3cm
SYBR Green Dye
SYBR Green dye is a fluorescent dye that binds to double-stranded DNA, but will stain single-stranded DNA with lower performance.
Single-Drop Fluorimeter
Fluorimeter is a device that is used to measure the amount fluorescence in a single drop.
How the Fluorescence Technique Works [In your own words, a summary of the information from page 9 of the worksheet]
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.
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.
Data Analysis
Representative Images of Samples
Image J Values for All Samples[See worksheet page 5]
BME 103 Spring 2013
.JPG)
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