Biomod/2011/TUM/TNT/Results: Difference between revisions

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<h3>FRET Measurement</h3>
<h3>FRET Measurement</h3>


We designed the structure in such a way that a small change of angle in the base, which is a 30 helix bundle in a honey comb lattice, is amplified by the two arms, which are both 10 helix bundles and therefore should twist as well. To measure the change in twist and angle two fluorophores were attached to the two arms so that a deformation should cause a change in distance between them. We chose a donor and an acceptor fluorophore, namely Atto 550 and Atto 647N, so a change in distance between them leads to a change in  FRET-efficiency.
A typical FRET-trace can be seen in the following video which also plots the donor, acceptor and FRET intenities over the time.
 
In order to [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Methods#Immobilization immobilize] our structure standing upright on the coverslide we used neutravidin and biotinylated oligos complementary to staples at the base of our structure, which is a common way to immobilize DNA origamis on surfaces.
 
To prepare the slides we used [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Immobilizing_structures_with_biotin-oligos_for_TIRF this] procedure.
 
 
The [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Methods#Fluorescence_Microscope_Setup fluorescence microscope] has three lasers with different wavelenghts (blue:473nm, green: 532nm, red: 640nm). We only used the red and the green one because of the dyes we attached to our “U”.
 
For the measurement we used alternating-laser excitation of single molecules (ALEX) with an excitation length of 0.05 sec.
A nice FRET-trace can be seen in the following video which also plots the donor, acceptor and FRET intenities over the time.


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<html><center><iframe width="425" height="349" src="http://www.youtube.com/embed/wyt8qzZpRog?hl=de&fs=1" frameborder="0" allowfullscreen></iframe></center></html>

Revision as of 20:35, 2 November 2011

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