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

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<h3>fluorescence analysis</h3>
<h4>FRET bulk measurements</h4>
We planned to measure bulk FRET at the photospectrometer and the RT-PCR. However, the photospectrometer is not sensitive enough to deal with Atto dyes at concentrations below 10 nM (peaks were not visible at all). The RT-PCR is more sensitive, fluorescence signals could be recorded from 50 µl samples with 10 nM Atto dyes. 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. To check the accuracy of the RT-PCR, measurements were set up in duplicate, i.e. 100 µl sample were prepared and subsequently divided into two 50 µl samples, making them exactly identical. However, reproducibility was poor, with deviations of up to 40% between identical samples. Due to this accuracy limitations set by the device, it was not reasonable to dilute the sample any further. However, the origami structure could only be applied in smaller concentrations because of the small folding batches. For this reason, we stopped experiments at the RT-PCR and instead went on with observations at the fluorescence microscope, thereby bypassing the problems of too small concentrations. <br>
<h4>FRET at the fluorescence microscope</h4>
<h4>fluorescence tracking at the fluorescence microscope</h4>




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