SYBR Green Dye
SYBR Green Dye is a molecular dye that fluoresces very well with dsDNA and weakly with water or single stranded DNA. When it binds to the DNA (preferably double-stranded though single stranded will still work) this dye absorbs blue light and emits green light. It is used to quantify and visualize double stranded DNA. When SYBR Green Dye is used with water there is no fluorescence. The more dsDNA the more the SYBR Green Dye emits the light.
Single-Drop Fluorometer
An instrument that detects florescence in a sample size of a single drop of 160 μL. We used a smart phone fluorimeter in this experiment. The Single-Drop Fluorometer consists of an apparatus that hold a specific slide, one with a dot grid present, and a simple box that covers it. A phone is set up in a stand so it does not need a hand to hold it and let light in while the picture is being taken. In the area of the apparatus which holds the slide there is a LED light which shines thru a drop placed over the middle two wells on the special slide. This LED light is controlled by a switch on the side of the apparatus.
How the Fluorescence Technique Works
The fluorescence technique works by having the dsDNA give a visual color signal for when it is present and having the fluorimeter capture a picture of the fluorescence that was produced. The concentration of DNA can be measured through this technique.
Procedure
Smart Phone Camera Settings
Type of Smartphone: Galaxy S3
Flash: Inactivated
ISO setting: 800
White Balance: Auto
Exposure: Highest Setting
Saturation: Highest Setting
Contrast: Lowest Setting
Calibration
We Placed our smart phone on the cradle at a right angle from the slide. We then adjusted the height of the fluorimeter using the plastic trays so that our camera was able to take a picture of the sample drop sideways.
Distance between the smart phone cradle and drop =7.0cm
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.
Using gloves, find the "smooth" side of the glass slide.
Turn on fluorimeter.
Place a slide in the fluorimeter with the smooth side facing down.
Set camera timer for 5 seconds. Calibrate the photo options and lay the smartphone on cradle with camera app on.
Adjust height of fluorimeter to get a camera view of the slide nearly edge-on by placing on top of a stable, supporting lid.
Place 80 uL drop of SYBR Green I solution on first 2 clear circles in middle of slide.
Changing tips, place 80 uL drop of sample/calibration solution on top of the SYBR GREEN I drop.
Adjust the slide so that the light illuminates the center of the drop and the drop focuses the light on the other side.
Adjust distance between smartphone and fluorimeter to 7cm away (>4cm).
Cover with light box but keep one flap up.
Make sure drop is focused one more time.
Depress the timer button on the camera to take the picture and lower the flap before picture is taken.
Remove the 160 uL drop using same tip from slide and discard liquid, as well as micropipetter tip, in waste liquid container.
Move the slide in the next position (center of next two circles). Once you have used all 5 possible measurement positions on the slide, switch slides.
Repeat steps 1-14 until all 8 samples have been photographed accordingly.
Data Analysis
Representative Images of Samples
The following images show two samples in he fluorometer. The first pictures shows the negative control, the one with no DNA. The second pictures shows the positive control, the one with DNA.
Image J Values for All Samples
PCR Product TUBE LABEL
Concentration
Volume of the DILUTED PCR Product solution (µL)
Volume of the SYBR GREEN I Dye solution (µL)
INTDENS VALUES BASED ON 3 SEPARATE DROP MEASUREMENTS
'
'
AVERAGE INTDENS VALUE
Corrected PCR Concentration
1. 2.5 Calf Thymus
2.25
80
80
7298707
5868419
6324719
6497281.7
27.00
2. 0.5 Calf Thymus
0.50
80
80
2282276
2152201
3068249
2500908.7
6.00
3. 0.25 Calf Thymus
0.25
80
80
1730534
2262858
2032111
2008501.0
3.00
4. 0 Calf Thymus
0.00
80
80
1464326
1440224
1654233
1519594.3
0.00
5. + (DNA)
1.77
80
80
4796701
4114399
4693591
4534897.0
21.21
6. P11 (DNA)
0.21
80
80
1310944
1260384
1673944
1415090.7
2.49
7. P12 (DNA)
0.59
80
80
1459086
3138435
1942214
2179911.7
7.08
8. P13 (DNA)
0.47
80
80
1793887
1849966
2158787
1934213.3
5.61
9. - (DNA)
0.26
80
80
1461537
2052869
1075174
1529860.0
3.18
10. P21 (DNA)
0.39
80
80
1915305
1464238
1974133
1784558.7
4.71
11. P22 (DNA)
0.14
80
80
1051004
1464473
1296561
1270679.3
1.62
12. P23 (DNA)
0.46
80
80
1927895
2326738
1528947
1927860.0
5.57
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 21.21 μg/mL
Your negative control PCR result was 3.18 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 90616 : The average corrected concentration of this patients DNA is 5.06 μg/mL. The following image shows an example of the patient's PCR sample in fluorometer.
Patient 91514 : The average corrected concentration of this patients DNA is 3.97 μg/mL. The following image shows an example of the patient's PCR sample in fluorometer.
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 90616 : This patient's results are less than the positive control, 16.15 μg/mL less, but they are higher than the negative control by 1.88 μg/mL. This patient is much closer to the negative control results therefore this patient has a negative test result.
Patient 91514 : This patient's results are less than the positive control by 17.24 μg/mL but higher than the negative control by 0.79 μg/mL. This patient is drastically closer to the negative control therefore this patient has a negative test result.
BME 100 Spring 2014
