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
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
0
219692
15600
204092
0
170238
9781
160457
0
224961
36206
188755
0.25
199972
16399
183573
0.25
212717
16604
196113
0.25
261838
51734
210104
0.5
308692
26366
282326
0.5
299687
28626
271061
0.5
250030
27130
222900
1
319367
25827
293540
1
316802
24964
291838
1
376998
54321
322677
2
395675
40796
354879
2
388219
41357
346862
2
376253
27314
348939
5
606708
68417
538291
5
606527
66757
539770
5
561030
30019
531011
Quick Calibration
Final DNA Concentration
Rawintden of the Drop
5
638902
5
605890
5
579459
2
329861
2
312824
2
336588
1
289773
1
256764
1
203170
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
1. A1
80
80
336320
296694
283014
305343
2. A2
80
80
169580
135032
144862
149825
3. B1
80
80
178665
181323
152036
170675
4. B2
80
80
155173
169683
181131
168662
5. B3
80
80
120320
132195
125157
125891
6. C1
80
80
378752
344187
383689
368876
7. C2
80
80
331931
368891
376694
359172
8. C3
80
80
351645
331268
338909
340607
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.
BME 100 Spring 2014
