SYBR green dye is used as a nucleic acid stain and binds to DNA. The result is a DNA-dye-complex that absorbs blue light and emits green light. This dye can also bind to single-stranded DNA and RNA with a lower performance.
Single-Drop Fluorimeter
A single-drop fluorimeter is made up of a sturdy black cover used to block out the light. There is a phone cradle used o hold the camera while taking pictures. Inside the black cover there is a platform to hold the slides. There is also an LED light placed around the slides. This light emits a blue light used to see the SYBR green dye.
How the Fluorescence Technique Works
The blue LED light shines on the DNA-dye samples. The amount of DNA in each sample is measured based on the concentration of the green light being emitted from the sample.
Procedure
Smart Phone Camera Settings
Type of Smartphone: iPhone 4s
Flash: off
ISO setting: N/A
White Balance: N/A
Exposure: N/A
Saturation: N/A
Contrast: N/A
Calibration
The camera was set on a cradle eye level with the sample. We set the camera with a timer so there would be no other light than the LED light the green glow coming from the sample. We clicked the button and closed the flap of the outer black case, then the camera took the picture of the sample.
Distance between the smart phone cradle and drop = 8 cm
Solutions Used for Calibration
Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL)
Volume of the 2X DNA solution (µL)
Volume of the SYBR GREEN I
Dye solution (µL)
Final DNA concentration in SYBR Green I solution (µg/mL)
5
80
80
2.5
2
80
80
1
1
80
80
0.5
0.5
80
80
0.25
0.25
80
80
0.125
0
80
80
0
Placing Samples onto the Fluorimeter
Place the fluorimeter into the box
Place the camera in the cradle
Adjust the height of the fluorimeter with the trays so the camera will see the drop at eye level
Insert the slide into the fluorimeter
Adjust the distance between the camera and the slide to be as close as possible without being blury
Place an 80 microliters drop of SYBR Green in between the first two rows of the slide
Add 80 microliters of the indicated DNA solution to the drop of SYBR Green
Align the sample and the blue LED light
Click the timer on the camera and close the lid for the picture
Take one more picture, for a total of two pictures, without moving the camera
Data Analysis
Representative Images of Samples
[Instructions: Show an IMAGE where you drew a circle around the droplet with the freehand tool for a sample with no DNA]
[Instructions: Show an IMAGE where you drew a circle around the droplet with the freehand tool for a sample with DNA (positive signal)]
[Instructions: See worksheet page 8. To save time on typing a new Wiki table from scratch, use THIS TOOL to auto-generate a Wiki table: Excel-to-Wiki Converter. Copy the headers and values from the Excel spreadsheet you made, paste them into the form field, click submit, copy the Wiki code that the tool generated, and replace TABLE GOES HERE (below) with your auto-generated code.]
BME 100 Spring 2014
