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Transcript 
The Photobleaching
Effect
by
Stephen Payne
?
SYBR Green
• Does not bind to single-stranded DNA
• Binds to double-stranded DNA and
fluoresces
LED
The Problem
Double-Stranded
DNA
Bleached
SYBR Green
Light
Fluorescent
SYBR Green
Suspicion of The Problem
Raw Data of Successive Runs of the Same 20 bp Sample
1.2
Photodiode Voltage (V)
First Run
Second Run
1
0.8
0.6
0.4
0.2
1.04
1.06
1.08
1.1
1.12
1.14
RTD Voltage (V)
1.16
1.18
1.2
Bleaching Function
Cold Sample Run with 4.3 Ohm Resistor (20 bp, 5mM KCl)
1
Raw Data
Exponential Fit
Relative Fluorescence
0.9
0.8
0.7
0.6
Flu = e
-.00078t
0.5
0.4
0
200
400
600
800
Time (seconds)
1000
1200
Bleaching Function
Cold Sample Run with 4.3 Ohm Resistor (20 bp, 50 mM KCl)
1
Relative Fluorescence
Raw Data
Exponential Fit
0.9
0.8
0.7
0.6
0.5
0
Flu = e
100
-.00068t
200
300
400
500
600
Time (seconds)
700
800
900
Correction by Bleaching Factor
Melting Curves for 20 bp, 5 mM KCl Sample
Normalized Photodiode Voltage
2
Uncorrected for Bleaching
Corrected for Bleaching
1.5
1
0.5
0
30
40
50
60
Temperature (°C)
70
80
Insignificant Bleaching Effect?
Cold Sample with 4.3 Ohm Resistor (20 bp, 100 mM KCl)
1
Relative Fluorescence
0.9995
0.999
0.9985
0.998
0.9975
0
200
400
600
800
1000 1200
Time (seconds)
1400
1600
1800
The Solution
• Change R1 in LED Driver from a
4.3 Ohm resistor to a 20 Ohm
resistor
Reduced Bleaching Effect
Cold Sample Run with 20 Ohm Resistor (20 bp, 5 mM KCl)
1.02
Relative Fluorescence
1
0.98
0.96
0.94
0.92
0.9
0.88
0.86
0.84
0
100
200
300
400
500
Time (seconds)
600
700
800
The Theory
20 Ohms
Signal
Optimal
Signal
4.3 Ohms
R optimal
Resistance (Ohms)
Conclusion
• Photobleaching significantly
affected our system with the 4.3
Ohm resistor
• This effect was reduced via
increasing the resistance in the
LED driver
• Further optimization of the
system is possible
Acknowledgements
Code
• Danielle Carpenter, Co-author
• Steve Wasserman, Debugging
Images
• http://distancelearning.ksi.edu/demo/bio378/DNA_files/i
mage023.jpg
• http://en.wikivisual.com/images/0/04/NA-comparedtoDNA_thymineAndUracilCorrected.png
• http://www.elixa.com/light/arrays2.jpg
• http://openwetware.org/wiki/20.309:Measuring_DNA_Me
lting_Curves
Questions?
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