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

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<h1>Fluorescence Measurements</h1>
<h1>Fluorescence measurements</h1>


<h2>FRET Bulk Measurements</h2>
<h2>FRET bulk measurements</h2>
For first tests, a simple [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Helix_MH_255/256 18 bp DNA double helix] with Atto 550 ddCTP at the one end and Atto 647N ddUTP at the other end was examined.
For first tests, a simple [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Helix_MH_255/256 18 bp DNA double helix] with Atto 550 ddCTP at the one end and Atto 647N ddUTP at the other end was examined.
The idea to perform bulk measurements based on FRET using a photospectrometer and a real time PCR was unsuccessful.  
The idea to perform bulk measurements based on FRET using a photospectrometer and a real time PCR was unsuccessful.  
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To handle the issue with the small concentrations further experiments were done with a fluorescence microscope.
To handle the issue with the small concentrations further experiments were done with a fluorescence microscope.


<h2>FRET at the Fluorescence Microscope</h2>
<h2>Single molecule measurements at the fluorescence microscope</h2>


 
<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.  
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.  
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<h3>Fluorescence Tracking at the Fluorescence Microscope</h3>
<h3>Fluorescence tracking</h3>


Besides FRET-measurements we also applied another approach to investigate the deformation of the structure where we determine the distance between the fluorophores and thereby get the distance of the two arms by directly comparing two images. At first we excite the Atto 550 dye and observe at its characteristic wavelength and then excite the Atto 647N dye and observe at its characteristic wavelength.
Besides FRET-measurements we also applied another approach to investigate the deformation of the structure where we determine the distance between the fluorophores and thereby get the distance of the two arms by directly comparing two images. At first we excite the Atto 550 dye and observe at its characteristic wavelength and then excite the Atto 647N dye and observe at its characteristic wavelength.

Revision as of 19:58, 2 November 2011

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