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OUR TEAM
LAB 5 WRITE-UP
Background Information
SYBR Green Dye
SYBR green dye is a cyanide fluorescent dye that binds to double stranded DNA. The DNA dye complex absorbs blue light and emits green light. The stain binds to double stranded DNA. SYBR Green has many uses especially in biochemistry, and molecular genetics areas. In PCR it is used for the quantification of double stranded DNA. The detection of the DNA is modified by the increase in fluorescence over the cycle. This dye is not very mutagenic. Unlike other dyes, this dye can also be added directly to the DNA sample.
Single-Drop Fluorimeter
The fluorometer consists of a small black box that holds down a hydrophobic slide and emits a blue light. It takes a single drop of the SYBR green dye mixed with a sample of the DNA that is placed on to the rough side of the slide. This technique is used measures intensity, wavelength, and emission to verify concentrations. The blue LED light that is emitted from the device focuses on the drop causing the intensity of it to increase. Therefore, making the reading of the light more profound on the Image J program so it is easier to get. The single drop fluorometer can verify that there are certain concentrations of RNA, DNA, or protein in a sample through its measurements. This technique is widely used in labs.
Fluorescence Technique
The fluorimeter works by using the fluorescence technique. SYBR Green 1 dye is placed on the slide and then the DNA sample is placed on top of the drop of the dye. The SYBR Green 1 dye binds to the DNA present in the sample. When the blue light is shined on the sample, the SYBR green dye will glow according to the amount of DNA present. For example, if a large amount of DNA is present then it glow with a bright green. If there is a small amount of DNA, there will either be little to no green glow. Since it is hard to see all of the green light with the naked eye, a picture is taken of the sample and it is imported into ImageJ. The image is then separated into its color channels and the green channel is used. This allows for the measurement of green light without picking up additional color. Its important to use a positive and negative control in addition to the using the samples. This way there can be a baseline for positive and a baseline for negative. All of the RawIntDen values are compared to the positive and negative control values to determine if the samples were positive or negative.
Below is an image taken while using the flourimeter. This image shows that the light emitted is blue and the SYBR dye is glowing green.
Procedure
Smart Phone Camera Settings
- Type of Smartphone: iPhone 5
- Flash: Off
- ISO setting: N/A
- White Balance: N/A
- Exposure: N/A
- Saturation: N/A
- Contrast: N/A
- (The settings were set to automatic on the iPhone 5. The ISO setting, white balance, exposure, saturation, and contrast were not adjusted because it was not possible to adjust them using the iOS camera app.)
Calibration
In order to setup the fluorimeter, the fluorimeter was first turned on and placed on a flat surface. Once this was done, a slide with the rough side up was inserted into it. The smartphone was placed in the cradle and in such a way that you get a clear view of the drop on all three positions of the slide were obtained.
- Distance between the smart phone cradle and drop = 6 cm
Below is a picture of the set up.
Solutions Used for Calibration
| Initial Concentration of 2X Calf Thymus DNA solution (ug/mL) |
Volume of the 2X DNA solution (ul) |
Volume of the SYBR GREEN 1 Dye Solution (ul) |
Final DNA Concentration in SYBR Green I solution (ug/mL)
|
| 5 |
80 |
80 |
2.5
|
| 2.5 |
80 |
80 |
1
|
| 1 |
80 |
80 |
0.5
|
| 0.5 |
80 |
80 |
0.25
|
| 0.25 |
80 |
80 |
0.125
|
| 0 |
80 |
80 |
0
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Placing Samples onto the Fluorimeter
- Setup the fluorimeter with the cell phone in the cradle and as close as possible so that a picture of the first two rows of the slide will be in focus.
- Place the slide on the fluorimeter so that the rough, hydrophobic side is facing up and the blue light is emitting between the first two rows of dots.
- Fill the micropipette with 80 ul of SYBR GREEN 1 dye solution.
- Put the drop of SYBR GREEN 1 dye solution onto the slide in the middle of the first two rows on the slide.
- Replace the micropipette tip.
- Fill the micropipette with the 80 ul of the 2X Calf Thymus DNA solution. Start with initial concentration of 5 ug/mL.
- Put the drop of 2X Calf Thymus DNA solution onto the drop of SYBR GREEN 1 dye solution.
- Check to make sure that the blue light is emitting across the center of the drop.
Data Analysis
Representative Images of Samples
The below image shows a drop in the fluorimeter that does not have DNA. This image is showing the color green channel and the absence of lighter colors in drop indicates a lack of the SYBR Green 1 dye. This also indicated that there is no DNA in the drop.
The below image shows a drop in the fluorimeter that does have DNA. The image is shown in the color green channel and there is a lot of lighter colors indicating green light present. The green light present indicates the presence of SYBR Green 1 dye and DNA.
Image J Values for All Samples
Original Calibration
| Final DNA Concentration |
Rawintden of the Drop |
Rawintden of the Background |
Rawintden drop - Rawintden background
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| 0 |
219692 |
15600 |
204092
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| 0 |
170238 |
9781 |
160457
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| 0 |
224961 |
36206 |
188755
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| 0.25 |
199972 |
16399 |
183573
|
| 0.25 |
212717 |
16604 |
196113
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| 0.25 |
261838 |
51734 |
210104
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| 0.5 |
308692 |
26366 |
282326
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| 0.5 |
299687 |
28626 |
271061
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| 0.5 |
250030 |
27130 |
222900
|
| 1 |
319367 |
25827 |
293540
|
| 1 |
316802 |
24964 |
291838
|
| 1 |
376998 |
54321 |
322677
|
| 2 |
395675 |
40796 |
354879
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| 2 |
388219 |
41357 |
346862
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| 2 |
376253 |
27314 |
348939
|
| 5 |
606708 |
68417 |
538291
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| 5 |
606527 |
66757 |
539770
|
| 5 |
561030 |
30019 |
531011
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Quick Calibration
| Final DNA Concentration |
Rawintden of the Drop
|
| 5 |
638902
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| 5 |
605890
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| 5 |
579459
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| 2 |
329861
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| 2 |
312824
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| 2 |
336588
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| 1 |
289773
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| 1 |
256764
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| 1 |
203170
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PCR Product Sample Measurements
| PCR Product TUBE Label |
Volume of the DILUTED PCR Product solution (ul) |
Volume of the SYBR GREEN 1 Dye Solution (ul) |
INTDENS VALUES BASED ON 3 SEPARATE DROP MEASUREMENTS |
' |
' |
AVERAGE INTDENS VALUE
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| 1. A1 |
80 |
80 |
336320 |
296694 |
283014 |
305343
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| 2. A2 |
80 |
80 |
169580 |
135032 |
144862 |
149825
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| 3. B1 |
80 |
80 |
178665 |
181323 |
152036 |
170675
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| 4. B2 |
80 |
80 |
155173 |
169683 |
181131 |
168662
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| 5. B3 |
80 |
80 |
120320 |
132195 |
125157 |
125891
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| 6. C1 |
80 |
80 |
378752 |
344187 |
383689 |
368876
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| 7. C2 |
80 |
80 |
331931 |
368891 |
376694 |
359172
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| 8. C3 |
80 |
80 |
351645 |
331268 |
338909 |
340607
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Fitting a Straight Line
PCR Results Summary
Instructor's summary: You completed 8 PCR reactions in a previous lab. You used the SYBR Green 1 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.
- Our positive control PCR result was 0.1399 ug/mL.
- Our negative control PCR result was 0.003238 ug/mL.
- Patient ID 40908 was 0.01595, 0.0141, 0.002527 ug/mL. These full color images looked like there was no SYBR Green Dye 1. There was no green color, just the blue from the light. The green color channel image did not show any lighter areas in the drop.
- Patient ID 51602 was 0.1984, 0.1894, 0.1723 ug/mL. These full color images looked like there was a lot of SYBR Green Dye 1. There was a majority of the green color in the drop. The green color channel image showed the drop was saturated in a lighter color, indicated the presence of the SYBR Green Dye 1.
- When compared to the positive and negative controls, the patient ID 40908 results is considered to be negative. The reason the team came to this conclusion is because the PCR results were closely related to the negative control PCR results. The images were also similar and showed no SYBR Green Dye 1.
- When compared to the positive and negative controls, the patient ID 51602 results is considered to be positive. The reason the team came to this conclusions is because the PCR results were closely related to the positive control PCR results. The images were also similar and showed a saturations of SYBR Green Dye 1.
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BME 100 Spring 2014
