SYBR Green Dye
The SYBR Green Dye is a special florescent dye that was used to help sense the presence of an enzyme in a DNA sample. The dye is dependent on how strong the enzyme is to make the sample glow a bright green.
Single-Drop Fluorimeter
A Single-Drop Fluorimeter is a structure inside of another structure. The outer layer of the device is a shield like structure that is designed to keep the light out. The structure that is placed on the inside is composed of two components, a phone holder and the actual fluorimeter. The fluorimeter has a slot that allows for a glass slide with small indentations, referred to by the group as craters, to hold the drops in place upon shutting the device and capturing a picture of the DNA sample. There is a black light that makes the fluorescent enzyme show up as a green color instead of a clear blue.
How the Fluorescence Technique Works
The technique used for the fluorescence is brought out when the correct enzyme is in the DNA sample. For a clearer picture of the sample, the box was shut to enhance the quality of the photograph. A phone was place on the bench with the flash off and the timer was set to 10 seconds to have time to focus and sharpen for a better picture. Three pictures were taken for each sample to ensure that there was a clear picture. It was repeated for each sample 3 times, but on the next section of craters of the glass plate.
Procedure
Smart Phone Camera Settings
Type of Smartphone: iPhone 5s
Flash: Off
ISO setting: Auto
White Balance: Auto
Exposure: N/A
Saturation: Auto
Contrast: Auto
Calibration
The smartphone was placed in the cradle and adjusted so that it was positioned vertically from the slides beneath the fluorimeter. Then, the distance between the drop and the iPhone5s was measured and the camera was set to focus on the position of the drop. The lid was closed to block out as much possible light, while still allowing access to the camera button. Each drop was photographed three times to provide a wider selection of data and to correct for any outliers. Three images from each concentration were then analyzed using ImageJ by splitting the color channels and selecting the green color scale, drawing a precise oval around the drop and then measuring area, mean pixel value and the integrated density for the drop and the background. The resulting value determined by the following equation, Integrated Density of Drop-Integrated Density of Background, was plotted versus the concentration. The linear best fit line of this plot was then found using Excel's Best Fit function. This best fit line can be used in future measurements to plug in the concentration being used for the value "x" and to determine the calibrated integrated density "y".
Distance between the smart phone cradle and drop = 4.2cm
Solutions Used for Calibration
Calf Thymus DNA Solution Concentration (microg/ml)
Volume of the 2X DNA solution (uL)
Volume of SYBR Green I Dye Solution (uL)
Final Concentration SYBR Green assay (ng/ml)
5
80
80
2.5
2
80
80
1
1
80
80
.5
.5
80
80
.25
.25
80
80
.125
0
80
80
blank
Placing Samples onto the Fluorimeter
First, use the plastic pipette to place 80 micro liters of SYBR green onto a middle dot on the glass slide in the fluorimeter.
Dispose of used pipette tip.
Using the plastic pipette, place 80 micro liters of the sample solution onto the same dot the SYBR green occupies.
Next, adjust the fluorimeter so the blue led light is focused on the liquid drop.
Place the camera phone 4.2 cm from the fluorimeter.
Cover the fluorimeter with the light box.
Use the timer on the camera phone to take two pictures of the sample. Make sure the camera is focused before the images are taken.
Use the plastic pipette to remove the drop from the glass slide.
Repeat the above method three times for each concentration being evaluated. Make sure to change out pipette tips between each new use.
Data Analysis
Representative Images of Samples
Sample Images Prior to Being Imported and Analyzed in ImageJ:
Flourometer Sample with No DNA (0 μg/mL) Loaded into ImageJ, Green Portion with Droplet Selected
Flourometer Sample with Positive DNA (2.5 μg/mL) Loaded into ImageJ, Green Portion with Droplet Selected
BME 100 Fall 2013
