SYBR Green Dye
SYBR Green Dye is used as a nucleic acid stain; it binds to DNA and absorbs blue light wavelengths while emitting green light wavelengths. While fluorescing very well in the presence of sDNA (double-stranded DNA), RNA and single stranded DNA found in the ending solution of PCR is still highlighted, but not at such a high performance level. Water molecules are not highlighted at all; water containing SYBR Green Dye can be used as a negative control when testing the quantitative results of a PCR machine. Having such a consistent outcome makes SYBR Green Dye the best reagent to use when checking the efficiency of a PCR machine because only completely copied double strands of DNA will be highlight during the use of the fluorimeter (the machine that emits blue light). The brighter the green color of a drop when placed on a fluorimeter, the greater the number of double-stranded DNA present in the sample.
Single-Drop Fluorimeter
A fluorimeter is a device used to measure the intensity and wavelengths of fluoresce through the use of light. It has two detectors, one that measures how much light is absorbed and one that measures how much light is emitted. The fluorimeter itself is a black box that has an area on the top of it to place a glass slide with a sample and a horizontally placed blue LED light facing the placement of the slide (as shown below). The device includes a small box that has multiple wells and a glass plate where the sample is placed. There are two LED lights on either side of the sample that shine a blue LED through the sample.
Fluorimeter Set-Up
How the Fluorescence Technique Works
Fluorescence technique uses the emission of light due to a molecule's absorption of a shorter wavelength to create an image. In a Single-Drop Fluorimeter, blue light (wavelength value of ~475nm) is emitted from an LED in order to excite the molecules of the SYBR Green Dye that have bonded with the double-stranded DNA in the drop of solution taken from the PCR machine at the end of a completed cycle. Once the blue light is absorbed by the slide sample that has been placed on the fluorimeter, a green light is reflected by the sample (wavelength of ~510nm). This process is essentially what fluorescing is: absorbing one wavelength of which a solution is being exposed to and emitting another higher wavelength. Fluorimeters are intended for assisting in the detection of fluorescence.
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
Fluorimeter and Camera Setup
The camera was positioned in the cradle so that it was standing upright at a 90 degree angle. The cradle and camera is supposed to be at distance greater than 4 cm away from the fluorimeter. In this experiment, it was approximately 5.9 cm away. The camera was also level with drop and focused on it. During the experiment, a picture was taken every three seconds, for a total of three pictures per drop.
Distance between the smart phone cradle and drop = 5.9 cm
Solutions Used for Calibration[Instructions: See worksheet page 6.]
Calf Thymus DNA solution concentration (microg/mL)
Volume of the 2X DNA solution (µL)
Volume of the SYBR GREEN I Dye solution (µL)
Final DNA concentration in SYBR Green I Assay (ng/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
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[Add more rows as needed]
Placing Samples onto the Fluorimeter
Step one, insert the glass slide into the fluorimeter with the rough side facing up.
Step two, turn on the fluorimeter and adjust the slide so that the light emitted from the fluorimeter passes between two sets of holes.
Step three, use the micropipette to drop 80µL of SYBR GREEN I on the slide so that the light travels through the drop.
Step four, use the micropipette to add 80µL of the first of six different concentrations of the DNA solution to the drop of SYBR GREEN I.
Step five, use the micropipette to remove the liquid on the slide after the experiment.
Step six, adjust the slide in the fluorimeter so that its light passes between two new sets of holes.
Step seven, repeat steps three through six, but remember to change the concentration of the DNA solution.
BME 100 Fall 2013
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