The Photobleaching Effect

byStephen Payne

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SYBR Green

• Binds to double-stranded DNA and fluoresces

• Does not bind to single-stranded DNA

LED

Light

Fluorescent SYBR Green

Bleached SYBR Green

Double-Stranded DNA

The Problem

1.04 1.06 1.08 1.1 1.12 1.14 1.16 1.18 1.20.2

0.4

0.6

0.8

1

1.2Raw Data of Successive Runs of the Same 20 bp Sample

RTD Voltage (V)

Ph

oto

dio

de

Vo

ltag

e (

V)

First RunSecond Run

Suspicion of The Problem

0 200 400 600 800 1000 12000.4

0.5

0.6

0.7

0.8

0.9

1

Re

lativ

e F

luo

resc

en

ce

Time (seconds)

Cold Sample Run with 4.3 Ohm Resistor (20 bp, 5mM KCl)

Raw DataExponential Fit

Bleaching Function

Flu = e-.00078t

0 100 200 300 400 500 600 700 800 9000.5

0.6

0.7

0.8

0.9

1

Re

lativ

e F

luo

resc

en

ce

Time (seconds)

Cold Sample Run with 4.3 Ohm Resistor (20 bp, 50 mM KCl)

Raw DataExponential Fit

Bleaching Function

Flu = e-.00068t

30 40 50 60 70 800

0.5

1

1.5

2

Temperature (°C)

No

rma

lize

d P

ho

tod

iod

e V

olta

ge

Melting Curves for 20 bp, 5 mM KCl Sample

Uncorrected for BleachingCorrected for Bleaching

Correction by Bleaching Factor

0 200 400 600 800 1000 1200 1400 1600 18000.9975

0.998

0.9985

0.999

0.9995

1Cold Sample with 4.3 Ohm Resistor (20 bp, 100 mM KCl)

Time (seconds)

Re

lativ

e F

luo

resc

en

ceInsignificant Bleaching Effect?

The Solution• Change R1 in LED Driver from a

4.3 Ohm resistor to a 20 Ohm resistor

0 100 200 300 400 500 600 700 8000.84

0.86

0.88

0.9

0.92

0.94

0.96

0.98

1

1.02Cold Sample Run with 20 Ohm Resistor (20 bp, 5 mM KCl)

Time (seconds)

Re

lativ

e F

luo

resc

en

ceReduced Bleaching Effect

Resistance (Ohms)

Sig

nal

The Theory

OptimalSignal

R optimal

4.3 Ohms

20 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

AcknowledgementsCode• Danielle Carpenter, Co-author

• Steve Wasserman, Debugging

Images• http://distancelearning.ksi.edu/demo/bio378/DNA_files/

image023.jpg

• http://en.wikivisual.com/images/0/04/NA-comparedto-DNA_thymineAndUracilCorrected.png

• http://www.elixa.com/light/arrays2.jpg

• http://openwetware.org/wiki/20.309:Measuring_DNA_Melting_Curves

Questions?