SYBR Green Dye SYBR dye is a molecular dye that has fluorescence qualities in the presence of double stranded DNA but not in water or with single stranded DNA. This dye is used to show the presence of dsDNA.
Single-Drop Fluorimeter The device shines a LED light through a drop of solution sitting on a hydrophobic surface slide that causes the molecules to stick together instead of the plate a spherical shape.
How the Fluorescence Technique Works SYBR GREEN I dye is used because it fluoresces in the presence of double stranded DNA. When taking a picture of this the fluoresces is picked up the pixels can be analyzed.
Procedure
Smart Phone Camera Settings
Type of Smartphone: Droid Razor
Flash: Off
ISO setting: 800
White Balance: Auto
Exposure:highest
Saturation:highest
Contrast: lowest
Calibration
Distance between the smart phone cradle and drop = 6cm
Experimental Set Up
Solutions Used for Calibration '
Concentration of 2X Thymus DNA (micrograms/mL)
Volume of 2X DNA solution(uL)
Volume of the SYBR GREEN I soluion(uL)
Final DNA concentration in SYBR green I solution(ug/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
First using the micropipette, pipette 80 microliters of SYBR Green solution onto the slide in the middle on the first row. Then pipette 80 microliters of the 2X calf thymus DNA solution requested in the same place.
The blue LED light was focused on the sample on the plate, and positioned so it is in the middle of the black fiber optic.
The timer was Set on the Smartphone for 10 seconds being placing on the cradle (6cm away), then the lid was closed to the black box and allowing the picture to be taken in complete darkness. This was done twice for every sample
After the pictures were taken, the micropipette was used to remove the solution off the plate. This was repeated 3 times for every concentration.
Data Analysis
Representative Images of Samples Droplet with no DNA
Droplet with DNA
Image J Values for All Samples
Trial Number
Final DNA concentration in SYBR Green I solution
Area
Mean Pixel Value
RawintDen of the Drop
Rawintden of the background
INTDEN
1
0
94633
79.542
7527330
602362
6924968
1
0.25
118200
65.825
7780550
659701
7120849
1
0.5
107328
71.761
7701956
508440
7193516
1
1
115140
126.899
14611201
530473
14080728
1
2
102036
141.805
14469248
625685
13843563
1
5
98864
174.741
17769909
646348
17123561
2
0
122547
36.617
4487295
552944
3934351
2
0.25
122547
54.325
6657319
533710
6123609
2
0.5
122547
77.174
9457486
568182
8889304
2
1
122547
132.743
16267264
614826
15652438
2
2
122547
107.147
13130574
467618
12662956
2
5
122547
154.52
16813027
485789
16327238
3
0
109408
30.175
3301381
367028
2934353
3
0.25
109408
43.453
4754133
450131
4304002
3
0.5
109408
61.553
6734393
468917
6265476
3
1
109408
93.821
10264797
529161
9735636
3
2
109408
113.063
12369977
501564
11868413
3
5
109408
147.703
16159888
533696
15626192
Fitting a Straight Line
PCR Results Summary
PCR Product Tube Label
Average INTDENS value
PCR Product Concentration
Corrected PCR Product Concentration
Positive Control
2187513.667
0.910428461
10.92514153
541
172330
-8.873821385
-106.4858566
542
131309.3333
-9.072987574
-108.8758509
543
179070
-8.841096901
-106.0931628
Negative Control
154153
-8.962075528
-107.5449063
431
174222
-8.864635224
-106.3756227
432
174816
-8.861751197
-106.3410144
433
124983
-9.103703596
-109.2444431
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 concnetration of claf thymus DNA) to convert INTDEN values into DNA concentration. Your positive control and negative control samples are used to determine the threshold values for determining whether an unknown (patient) sample is truly positive or negative.
Your positive control PCR result was 10.9 μg/mL
Your negative control PCR result was -107.5 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 54174 : -107.2 μg/mL
The images were very dark. The droplets were transparent.
Patient 43184 : -107.3 μg/mL
The images were very dark. The droplets were transparent.
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 54174 : Negative because the patients results had an average of -107.2 μg/mL while the negative control had a value of -107.5 μg/mL and the positive control had a value of 10.9 μg/mL. Therefore the patient's results were much more similar to the negative control than the positive control.
Patient 43184 : Negative because the patient results had an average of -107.3 μg/mL while the negative control had a value of -107.5 μg/mL and the positive control had a value of 10.9 μg/mL. Therefore the patient's results were much more similar to the negative control than the positive control.