Biomod/2011/TUM/TNT/LabbookA/2011/08/11: Difference between revisions

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==measured absorption and emission spectra of Atto 550 and Atto 647N together with DNA binders==
==measured absorption and emission spectra of Atto 550 and Atto 647N together with DNA binders==
* 100nM Atto 550 ddCTP resp.100nM Atto 647N DUTP
100nM Atto 550 ddCTP resp.100nM Atto 647N dUTP were analyzed <br>
* DNA binders were added in a concentration according to 10 times their K<sub>D</sub> for binding DNA
DNA binders were added in a concentration according to 10 times their 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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* Ethidium bromide: K<sub>D</sub> = 12µM
* Ethidium bromide: K<sub>D</sub> = 12µM
* Methyl green:    K<sub>D</sub> = 2.2nM
* Methyl green:    K<sub>D</sub> = 2.2nM
<br>
buffer: 0.5x TBE, 11mM MgCl<sub>2</sub> <br>
sample volume: 100µl <br>
room temperature <br>
slit width: 5nm <br>
<br>
Atto 550: <br>
* excitation at 500nm and detection at 520-700nm
* excitation at 450-600nm and detection at 610nm
Atto647N: <br>
* excitation at 600nm and detection at 610-800nm
* excitation at 500-680nm and detection at 690nm
<br>
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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Revision as of 06:50, 7 September 2011

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measured absorption and emission spectra of Atto 550 and Atto 647N together with 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


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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