SYBR Green Dye SYBR Green Dye is a cyanine dye used in many molecular biology and biochemistry experiments as a nucleic acid stain. The dye is able to detect polymerase chain reactions products by binding to DNA by absorbing the blue light and emitting the green light, binding to DNA that is double stranded but still having the capability to stain single-stranded DNA and RNA. The PCR products are created and during the PCR the DNA polymerase amplifies the target sequence where the dye then binds to every new copy of double-stranded DNA. As the process continues along, more PCR product is created. One of them main disadvantages to the dye is the fact that the dye binds to strands that are nonspecific double-stranded DNA, producing what is known as a false positive meaning that you need to ensure the product is a well-designed primer for the best results with the DNA. Another problem we saw with the dye was that the color seemed to have "washed out" from the lighting in the room we were working with so we did not see any results until we switched to a different dye.
Single-Drop Fluorimeter A Single-Drop Fluorimeter device is a black box with a small slit at the top where a glass slide can fit into it. A sample can be placed on this glass slide where a blue LED light will shine onto the sample. The device then has a big black box to keep out the light from entering from the slides, and then a photo can be taken of the sample. The fluorimeter works by using detectors to measure the wavelengths of fluorescence through the use of light, measuring the light absorbed and the light emitted.
How the Fluorescence Technique Works When beginning the experiment, make sure the hydrophobic side of the slide is face up and place a drop of the dye on that side of the slide in the fluorimeter. The dye clumps together, forming one droplet on the slide and after adding the DNA to the droplet, the dye should exhibit a florescent appearance. The fluorescence is visible because the dye is placed on the slide where outside lighting is eliminated and instead, uses the light beams from the slide. A short wavelength is emitted from this fluorescent, producing photons. From these photons, the energy released can determine the samples' concentrations. The results can then be analyzed by the fluorescence in the pictures taken and we will then be able to identify the directly proportional relationship of the DNA and fluorescence.
Procedure
Smart Phone Camera Settings
Type of Smartphone: Samsung Galaxy S4
Flash: off
ISO setting: 800
White Balance: Auto
Exposure:+2.00
Saturation:N/A
Contrast:N/A
Calibration
In order to calibrate the phone to the fluorimeter, we used the guess and check method. Due to a lack of plastic plates to raise the fluorimeter, we used a box that at one point contained tips for the micropipette. We then elevated it further with a plastic plate. The phone cradle was also raised by a plastic plate, in order for us to get the right angle for the side profile shot of the drop. Therefore, the final set up was the fluorimeter on a plastic box stacked with a plastic plate, and the phone cradle slightly elevated by a plastic plate.
Distance between the smart phone cradle and drop = 12 cm
Solutions Used for Calibration
Calf Thymus DNA Solution Concentration (microg/mL)
Volume of the DNA Solution (uL)
Volume of the SYBR Green I Dye Solution (uL)
Final DNA Concentration in SYBR Green I Assay (ng/mL)
5
80
80
2.5
2
80
80
1
1
80
80
0.5
.5
80
80
0.25
.25
80
80
0.125
0
80
80
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Placing Samples onto the Fluorimeter
Place the slide smooth side down in the fluorimeter
Adjust the device so that the camera, when placed in the cradle, would be able to take a photo of the drop from a side view
Turn on the light using the switch on the side of the device
Put 80 uL of SYBR Green I solution, placed in between the two first dots in the middle column
Add 80 uL of the sample/calibration solution to the 80 uL of SYBR Green I, so that there is a drop with a total volume of 160 uL
Center the light in the middle of the drop and place the cradle 12 cm away from the center of the drop
Take 3 pictures per drop and repeat the steps again for another sample of the same concentration to total 2 trials with 3 pictures each.
Repeat steps 4-8 for each of the 6 concentrations: 0, .25, .5, 1, 2, and 5.
BME 100 Fall 2013
