Biomod/2011/TUM/TNT/LabbookA/2011/08/11

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<h2>Measured Absorption and Emission Spectra of Atto 550 & Atto 647N & with DNA Binders</h2>
<h2>Measured Absorption and Emission Spectra of Atto 550 & Atto 647N & with DNA Binders</h2>
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Many DNA binders interact with DNA via aromatic systems, or otherwise absorb in the range of UV/VIS light. This could cause background signals, which would impair the FRET measurements. Therefore, we recorded spectra of the Atto dyes mixed with the different DNA binders. <br>
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<br>
100nM Atto 550 ddCTP resp.100nM Atto 647N dUTP were analyzed <br>
100nM Atto 550 ddCTP resp.100nM Atto 647N dUTP were analyzed <br>
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DNA binders were added in a concentration according to 10 times their K<sub>D</sub> for DNA binding <br>
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DNA binders were added in a concentration according to 10 times their [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Glossary#DNA_Binders K<sub>D</sub>] for DNA binding <br>
* Hoechst 33258:    K<sub>D</sub> = 2.2nM
* Hoechst 33258:    K<sub>D</sub> = 2.2nM
* DAPI:            K<sub>D</sub> = 1.8nM
* DAPI:            K<sub>D</sub> = 1.8nM
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buffer: 0.5x TBE, 11 mM MgCl<sub>2</sub>; slit width: 5 nm; room temperature <br>
buffer: 0.5x TBE, 11 mM MgCl<sub>2</sub>; slit width: 5 nm; room temperature <br>
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Methyl green seems to interfere with absorption of Atto 550 and fluorescence of Atto647N. Ethidium bromide displays absorption and emission at similar wavelengths as Atto 550, but the spectra are additive, so the photophysical properties of Atto 550 are not altered by the presence of ethidium bromide. All other tested DNA binders do not affect Atto 550 as well.  
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Methyl green seems to interfere with absorption of Atto 550 and fluorescence of Atto647N. Ethidium bromide displays absorption and emission at similar wavelengths as Atto 550, but the spectra are additive, so the photophysical properties of Atto 550 are not altered by the presence of ethidium bromide. All other tested DNA binders do not affect Atto 550 as well. <br>
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We decided to concentrate on three DNA binding compounds, ethidium bromide as important example for an intercalator, DAPI as a minor groove binder and spermine as a major groove binder.  
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11th Aug 2011

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Contents

Measured Absorption and Emission Spectra of Atto 550 & Atto 647N & with DNA Binders

Many DNA binders interact with DNA via aromatic systems, or otherwise absorb in the range of UV/VIS light. This could cause background signals, which would impair the FRET measurements. Therefore, we recorded spectra of the Atto dyes mixed with the different DNA binders.

100nM Atto 550 ddCTP resp.100nM Atto 647N dUTP were analyzed
DNA binders were added in a concentration according to 10 times their KD for DNA binding

  • Hoechst 33258: KD = 2.2nM
  • DAPI: KD = 1.8nM
  • Spermine: KD = 4.8µM
  • Ethidium bromide: KD = 12µM
  • Methyl green: KD = 2.2nM


buffer: 0.5x TBE, 11mM MgCl2
sample volume: 100µl
room temperature
slit width: 5nm

Atto 550:

  • excitation at 500nm and detection at 520-700nm
  • excitation at 450-600nm and detection at 610nm

Atto647N:

  • excitation at 600nm and detection at 610-800nm
  • excitation at 500-680nm and detection at 690nm


Fig.1: Excitation and emission spectra of Atto 550 ddCTP resp. Atto 647N dUTP with and without different DNA binders
Atto dyes: 100 nM, DNA binders were added in a concentration according to 10x their KD (see references), i.e.: Hoechst 33258: 22 nM, DAPI: 18 nM, spermine: 48 µM, methyl green: 22 nM, ethidium bromide: 120 µM
EtBr without Atto 550 ddCTP refers to a spectrum with only 120 µM ethidium bromide, while Atto 550 ddCTP corrected for EtBr is the difference spectrum between ethidium bromide and EtBr without Atto 550 ddCTP
buffer: 0.5x TBE, 11 mM MgCl2; slit width: 5 nm; room temperature

Methyl green seems to interfere with absorption of Atto 550 and fluorescence of Atto647N. Ethidium bromide displays absorption and emission at similar wavelengths as Atto 550, but the spectra are additive, so the photophysical properties of Atto 550 are not altered by the presence of ethidium bromide. All other tested DNA binders do not affect Atto 550 as well.
We decided to concentrate on three DNA binding compounds, ethidium bromide as important example for an intercalator, DAPI as a minor groove binder and spermine as a major groove binder.