Biomod/2011/TUM/TNT/Results

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Depending on the background we decided to use the microscope either in epifluorescence or in TIRF modus.
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The analysis program is a [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Methods#Evaluation_of_Data matlab script] which searches for spots in the red and the green movie and plots the intensities over time to identify bleaching events. Only those plots where the acceptor bleaches first and the donor bleaches afterwards are useful to calculate the FRET-efficiency (see figure 8).  
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The analysis program is a [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Methods#Evaluation_of_Data matlab script] which searches for spots in the red and the green movie and plots the intensities over time to identify bleaching events. Only those plots where the acceptor bleaches first and the donor bleaches afterwards are useful to calculate the FRET-efficiency.
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[[Image:Schöner_FRET_Verlauf.PNG|x350px|thumb|Fig. 8 Intensities of donor and acceptor]]<br>
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[[Image:Schöner_FRET_Verlauf.PNG]]<br>
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<font size="1">'''Fig: Example of an intensity over time plot of the acceptor and donor'''</font>
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The graph shows the intensities of the donor and the acceptor and in addition the intensity of the FRET-events. As one can see the intensity of the donor rises as soon as the acceptor bleaches. After some while the donor bleaches too. From that the FRET-efficiency can be calculated.
The graph shows the intensities of the donor and the acceptor and in addition the intensity of the FRET-events. As one can see the intensity of the donor rises as soon as the acceptor bleaches. After some while the donor bleaches too. From that the FRET-efficiency can be calculated.
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We at first measured the FRET-efficiencies for the BM14 structure without any intercalator or groove binder as a control and afterward we measured the same structure with 4.8µM spermine. We plotted the FRET-efficiencies in the following histograms.
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First we measured the FRET-efficiencies for the [http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/Structure_page#BM12_to_BM14:_Fluorophore-labeled_Structures_for_FRET_Measurements.2C_including_Adapters_for_Immobilisation BM14 structure] without any intercalator or groove binder as a control and afterward we measured the same structure with 4.8µM spermine (corresponding to one molecule every 7bp). The FRET-efficiencies were plotted in figure 9.
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[[Image:BM14_control.PNG]]<br>
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[[Image:BM14_control.PNG|x350px|thumb|Fig. 9a Histogram of FRET efficiencies; negative control]]<br>
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<font size="1">'''Fig: BM14_control
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'''</font>
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[[Image:BM14_Spermin.PNG]]<br>
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[[Image:BM14_Spermin.PNG|x350px|thumb|Fig. 9b Histogram of FRET efficiencies; with 4.8µM spermine]]<br>
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<font size="1">'''Fig: BM14_spermine_4.8µM
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'''</font>
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It is obvious that we actually measured FRET, though the low yield of FRET-events that were found by the matlab script does not allow to draw any conclusions because of the low statistics. This could mean that there are not all of the staples were labeled correctly so that there are structures that only contain one fluorophore or even none.
 
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Yet the fact that there actually were FRET-events makes it worth to keep on elaborating these measurements.
 
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It is obvious that we actually measured FRET, though the low yield of FRET-events that were found by the matlab script does not allow to draw any conclusions because of the low statistics. The wide spread of FRET efficiencies is probably caused by the base twists observed in the TEM measurements. Here further optimization needs to be done. Yet the fact that there actually were FRET-events makes it worth to keep on elaborating these measurements.
<h3>Fluorescence Tracking</h3>
<h3>Fluorescence Tracking</h3>

Revision as of 23:48, 2 November 2011

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