Biomod/2011/TUM/TNT/LabbookA/2011/08/11: Difference between revisions
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* excitation at 500-680nm and detection at 690nm | * excitation at 500-680nm and detection at 690nm | ||
<br> | <br> | ||
<p>[[Image:Control spectra.png|490px]]</p> | |||
<font size="1"> | |||
<b> | |||
Fig.1: Excitation and emission spectra of Atto 550 ddCTP resp. Atto 647N dUTP with and without different DNA binders </b> <br> | |||
Atto dyes: 100 nM, DNA binders were added in a concentration according to 10x their K<sub>D</sub> (see [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Extras/References references]), i.e.: Hoechst 33258: 22 nM, DAPI: 18 nM, spermine: 48 µM, methyl green: 22 nM, ethidium bromide: 120 µM <br> | |||
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 <br> | |||
buffer: 0.5x TBE, 11 mM MgCl<sub>2</sub>; slit width: 5 nm; room temperature <br> | |||
</font size> <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. | 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. | ||
</div> | </div> | ||
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11th Aug 2011Measured absorption and emission spectra of Atto 550 and Atto 647N together with DNA binders100nM Atto 550 ddCTP resp.100nM Atto 647N dUTP were analyzed
Atto647N:
Fig.1: Excitation and emission spectra of Atto 550 ddCTP resp. Atto 647N dUTP with and without different DNA binders |