SYBR Green Dye
SYBR green; nucleic acis gel, is one of the most sensetive stains available for detecting double stranded DNA in agarose and polyacrylamide gels. because of its senseitivity, it is very useful for assays where the presence of contaminating RNA or ssDNA might obscure results. its easy to use as gels soaked in diluted stain can be visualized without desalting. it is detectable with UV lights, gel documentation systems and laser scanners. Severa other features make SYBR Green particularly useful as a nucleic acid stain. It is known to be stable over a range of temperatures, which means that it can be used to monitor biochemical reactions that take place at high and low temperatures. It bind non covelantly to the surface of the DNA, meaning that it will not interfere with the activity of most nucleases and DNA polymerases. It also binds to nucleic acids over a dynamic linear range of up to four orders of magnitude, giving an accurate quantification of DNA over a wide range of concentrations.
Single-Drop Fluorimeter
fluorimeter is a fluroscence parameter messurment device. its intensity and wavelength distribution of emission spectrum after excitation by a certain spectrum of light. these parameters are used to identify . these parameter are used to identify the presence and the amount of a certain molucule in the medium. they can detect dlurocent molcule concetration up to 1 ppt ( part per trillion) .
The design of any typical fluorometer has several key components. It has an input source for ordinary visible light, and this light is passed through an excitation filter that allows only specific wavelengths of it to impact onto a sample cell of the material being studied. When this material, whether organic or inorganic, is bombarded by these controlled wavelengths of light, it fluoresces, emitting characteristic light of its own that is then passed through an emission filter. The emissions are read by a light detector which produces a readout for the observer to know how the sample is reacting and what its contents are.
- Fluorimeter Set-Up
How the Fluorescence Technique Works
Fluorescence is a very sensitive detection technique. A beam of light shines through the drop at a certain wavelength and excites the electrons in molecules of the drop causing it to emit a light of a longer wavelength. The slide we are using is coated with a rough layer of Teflon but has circles of bare glass. This combination of properties results in a superhydrophobic surface that allows the solution to cling to the glass but repel from the layer of Teflon. The environment from the slide allows the blue LED light to focus on the drop and thus increases the intensity of the fluorescence event. The combination of the SYBR Green Dye I and the solution form a complex with the DNA that prefers to go to the surface creating the beach ball shaped drop on the slide. This specific material creates the perfect environment to then measure the fluorescence of the sample. In this particular experiment, we use ImageJ software to measure and analyze the degree at which the fluorescence is emitted from the sample to understand the components of the DNA sample.
Procedure
Smart Phone Camera Settings
Type of Smartphone: iPhone 4s
Flash: Off
ISO setting: Auto
White Balance: Auto
Exposure: Auto
Saturation: Auto
Contrast: Auto
Calibration
Turn the fluorimiter light on.
Adjust the camera's settings to match the above.
Place the phone on the cradle or stand at a right angle from the slide, perpendicular to the surface.
Adjust the height of the fluorimeter using the plastic trays so that the camera picture has a clear view of the side of the drop.
The set up of the fluorimeter should look similar to shown(right image) and when looking through the phone's camera screen, it should appear similar to shown (left image).
Distance between the smart phone cradle and drop = 8cm
Solutions Used for Calibration
Concentration of Calf Thymus DNA (µg/mL)
Volume of DNA Solution (µL)
Volume of SYBR Green I Dye Solution (µL)
Final Concentration of DNA in 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
1. Insert the slide into the fluorimiter with the smooth, glass side down.
2. Get a 80 microliter measurement of the SYBR Green I and place this drop in the middle of the first two rows of the slide. The drop should look like a ball on top of the slide.
3. Get a 80 microliter measurement of the calf thymus solutions and place this solution on top of the SYBR Green I.
4. Align the slide so that the blue LED light is focused by the drop to the middle of the black fiber optic fitting on the other side of the drop.
5. Set the camera timer, then put the light box over the whole setup.
Data Analysis
Representative Images of Samples
No DNA:
DNA:
Image J Values for All Samples
Final DNA concentration in SYBR Green I solution (µg/mL)
AREA
Mean Pixel Value
RAWINTDEN OF THE DROP
RAWINTDEN OF THE BACKGROUND
DROP-BACKGROUND
2.5
47600
175.604
8358740
192210
8166530
2.5
49624
173.63
8616223
209182
8407041
2.5
49624
176217
8744600
211479
8533121
1
49624
117.528
5832208
188774
5643434
1
49624
118.981
5904302
190959
5713343
1
49624
113.687
5641590
188851
5452739
0.5
49624
80.465
3992971
181507
3811464
0.5
49624
78.058
3873566
185690
3687876
0.5
49624
78.988
3919689
184420
3735269
0.25
49624
81.546
4046657
162423
3884234
0.25
49624
82.679
4102843
168964
3933879
0.25
49624
90.321
4482073
167377
4314696
0.125
49624
20.713
1027841
165444
862397
0.125
49624
21.425
1063195
163794
899401
0.125
49624
20.658
1025137
162862
862275
0
49624
7.155
355061
158941
196120
0
49624
6.243
309800
161363
148437
0
49624
6.479
321531
166295
155236
Fitting a Straight Line
PCR Results Summary
Instructor's summary: You completed 8 PCR reactions in a previous lab. You used the SYBR Green I staining and imaging technique to measure the amount of amplified DNA in each PCR reaction.
You used a standard curve (based on known concentrations of calf thymus DNA) to convert INTDEN values into DNA concentration. Your positive control and negative control samples should be used
as threshold values for determining whether an unknown (patient) sample is truly positive or negative.
Your positive control PCR result was 0.274126694 μg/mL
Your negative control PCR result was -0.041063194 μg/mL
Write-in each patient ID and give both a qualitative (what the images looked like) and a quantitative description (μg/mL) of what you observed
Patient 43849: None of the images contained any fluorescence. The samples for this patient had an average corrected concentration of -0.05994057 μg/mL
Patient 80853: All of the images contained fluorescence. The samples for this patient had an average corrected concentration of 0.293841144 μg/mL
Compare each patient's results to the positive control value and the negative control value. Draw a final conclusion for each patient (positive or negative) and explain why you made that conclusion.
Patient 43849: Because none of the images contained fluorescence and the samples came back negative, this patient must be the negative one.
Patient 80853: Because all of the images contained fluorescence and the samples came back positive, this patient must be the positive one.
BME 100 Spring 2014
