SYBR Green Dye is a dye that stains nucleic acids by binding to DNA or RNA. The dye is used to visualize DNA through staining agarose gels,
biochip applications, fluorescence imaging techniques, real-time PCR, and more. SYBR dye fluoresces when combined with dsDNA (Zipper Hubert Brunner 1). However, unbound SYBR Green is unreactive to light. This feature allows scientists to determine the concentration of a target DNA sample by measuring the intensity of the fluorescent light being emitted by the sample. The higher the intensity of the light being emitted, the more concentrated the sample is with the target DNA. SYBR Green, as its name implies, fluoresces with a green color. Accordingly, it is important to use a different wavelength of light to excite the sample in order to prevent distorting the results. There are several types of fluorescent dyes similar to the SYBR Green used in this lab, but SYBR Green was chosen for its environmental friendliness and high level of light emission intensity to allow for easier detection of binding to the target DNA.
Single-Drop Fluorimeter
A fluorimeter is a device used to measure and identify fluorescence in a medium. Fluorimeters measure certain wavelengths of light and have two light detectors, one of which detects absorbency while the other detects fluorescence emission (So Dong 1). Single-Drop Fluorimeters do this with a single drop of a sample that is placed on a hydrophobic slide in the path of a beam of single wavelength light. For example, in our setup we used blue LED light to allow for light absorption and prevent contaminated intensity data that would occur if we used a green LED since the SYBR Green Dye fluoresces in the green range. A light detector or camera placed at a uniform distance from this sample is then used to capture the intensity of the fluorescence being emitted from the sample. Cameras are acceptable for this laboratory set-up even though they are unable to measure light intensity because it is assumed that light intensity is proportional to brightness; the brighter an object is in a photo, the higher the light intensity.
(Figure 1: Our Single-Drop Fluorimeter setup. A sample is placed in the path of the LED light on the white hydrophobic slide and its light intensity is captured by taking a picture with the iPhone. Image by Nathan Kirkpatrick)
(Figure 2: Our Single-Drop Fluorimeter covered by a box to prevent ambient light interference. A timer is used to take the picture of the sample once the box is closed. Image by Nathan Kirkpatrick)
How the Fluorescence Technique Works
In this experiment, the concentration of DNA was determined by measuring the amount of fluorescence in a medium which had been dyed with SYBR Green Dye. Since light intensity is assumed to be proportional to brightness, images taken with a camera using the Single-Drop Fluorimeter setup described above can be used to determine the intensity of the SYBR Green Dye and, accordingly, the concentration of target DNA in the sample.
After a single-drop fluorimeter was used to measure the fluorescence and an image was taken, ImageJ software was used to determine the concentration of DNA.
Citations:
Zipper, Hubert, Herwig Brunner, Jurgen Bernhagen, and Frank Vitzthum. "Investigations on DNA Intercalation and Surface Binding by SYBR Green I, Its Structure Determination and Methodological Implications." Ncbi.nih.gov. US National Library of Medicine, 12 July 2004. Web. 3 Apr. 2014.
So, Peter TC, and Chen Y. Dong. "Fluorescence Spectrophotometry." Scribd. Macmillan Publishers Ltd, Nature Publishing Group, 16 Jan. 2009. Web. 03 Apr. 2014.
Procedure
Smart Phone Camera Settings
Type of Smartphone: Apple iPhone5S
Flash: no flash
ISO setting: 2500
White Balance: Auto
Exposure: Highest Setting
Saturation: N/A
Contrast: N/A
Calibration
[Instructions: Describe how to set up your camera in front of the fluorimeter. Add a PHOTO of this set-up for bonus points.]
Distance between the smart phone cradle and drop = 8cm
[Instructions: See worksheet page 6.]
Solutions Used for Calibration[Instructions: See worksheet page 6.]
row 1 cell 1
row 1 cell 2
row 1 cell 3
row 1 cell 4
row 2 cell 1
row 2 cell 2
row 2 cell 3
row 2 cell 4
row 3 cell 1
row 3 cell 2
row 3 cell 3
row 3 cell 4
[Add more rows as needed]
Placing Samples onto the Fluorimeter
[Instructions: Step one, in your OWN words]
[Instructions: Step two, in your own words]
[Instructions: Step three, in your own words]
[Instructions: Step etc., in your own words]
Data Analysis
Representative Images of Samples
(Figure 3: ImageJ Green-Channel isolated Negative Control image with circle around droplet)
(Figure 4: ImageJ Green-Channel isolated Positive Control image with circle around droplet)
Image J Values for All Samples
[Instructions: See worksheet page 8. To save time on typing a new Wiki table from scratch, use THIS TOOL to auto-generate a Wiki table: Excel-to-Wiki Converter. Copy the headers and values from the Excel spreadsheet you made, paste them into the form field, click submit, copy the Wiki code that the tool generated, and replace TABLE GOES HERE (below) with your auto-generated code.]
PCR Product Tube Label
Area (pixel^2)
Mean Pixel Value
RawIntDen of the Drop
PC 1
30528
123.781
3778777
PC 2
28794
124.966
3598262
PC 3
28349
126.204
3577769
NC 1
26035
49.289
1283252
NC 2
25986
38.364
1002239
NC 3
24081
34.513
831118
1R1 1
26780
40.389
1081609
1R1 2
28678
38.826
1113459
1R1 3
27348
39.13
1070129
1R2 1
27328
43.119
1178362
1R2 2
29178
45.662
1332320
1R2 3
26307
47.495
1249444
1R3 1
22214
44.459
987619
1R3 2
26264
32.101
843096
1R3 3
25368
33.248
843428
2R1 1
22296
126.872
2828741
2R1 2
25085
130.523
3274161
2R1 3
24628
130.723
3219456
2R2 1
26100
129.338
3375712
2R2 2
26272
128.394
3373160
2R2 3
23711
129.676
3074751
2R3 1
23900
128.988
3082802
2R3 2
23824
131.695
3137491
2R3 3
27428
131.43
3604871
PCR Product Tube Label
Average RawIntDen of the Drop
Average PCR Product Concentration μg/mL
Corrected Average PCR Product Concentration, μg/mL
PC
3651602.667
1.651602667
19.819232
NC
1038869.667
-0.961130333
-11.533564
1R1
1088399
-0.911601
-10.939212
1R2
1253375.333
-0.746624667
-8.959496
1R3
891381
-1.108619
-13.303428
2R1
3107452.667
1.107452667
13.289432
2R2
3274541
1.274541
15.294492
2R3
3275054.667
1.275054667
15.300656
Sample
Corrected PCR Product Concentration (μg/ml)
Postive Control
19.819232
Negative Control
-11.533564
Patent: 21489 Replicate 1
-10.939212
Patent: 21489 Replicate 2
-8.959496
Patent: 21489 Replicate 3
-13.303428
Patent: 90434 Replicate 1
13.289432
Patent: 90434 Replicate 2
15.294492
Patent: 90434 Replicate 3
15.300656
Fitting a Straight Line
[Instructions: Place an IMAGE of your Excel plot with a line of best fit here. See worksheet page 9]
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 19.819232 μg/mL
Your negative control PCR result was -11.533564 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 21489 : The samples containing this patient's PCR-duplicated DNA with SYBR Green did not appear to exhibit SYBR Green-affiliated fluorescence. In other words, the droplet did not glow at all. The Average Corrected PCR Product Concentration of -11.06737867 μg/mL for all three replicates of this patient supports this visual observation.
Patient 90434 :The samples containing this patient's PCR-duplicated DNA with SYBR Green did appear to exhibit SYBR Green-affiliated fluorescence. In other words, the droplet was visually glowing. The Average Corrected PCR Product Concentration of 14.62819333 μg/mL for all three replicates of this patient supports this visual observation.
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 21489 : The Average Corrected PCR Product Concentration of -11.06737867 μg/mL for all three replicates of this patient is very close to the negative control threshold value of -11.533564 μg/mL. From this, we can conclude that Patient 21489 does not contain the DNA sequence in question. We came to this conclusion because the SYBR Green fluoresced as much in this patient's samples as it did in the negative control where no target DNA sequences were present.
Patient 90434 :The Average Corrected PCR Product Concentration of 14.62819333 μg/mL for all three replicates of this patient is very close to the positive control threshold value of 19.819232 μg/mL. From this, we can conclude that Patient 21489 does contain the DNA sequence in question. We came to this conclusion because the SYBR Green fluoresced as much in this patient's samples as it did in the positive control where a significant amount of target DNA sequences were present. Unfortunately, Patient 90434 has cancer.