Biomod/2011/TUM/TNT/LabbookA/2011/09/13

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13th Sep 2011

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Fluorescence Microscopic Ensemble Measurement of BM5_5/20

The concentration of labeled DNA was too low for measurements at the real rime PCR or photospectrometer.

Therefore, a new method is tried to measure the FRET efficiency of the U structure BM5_5/20: ensemble measurement in a fluorescence microscope


After filtering the structure to remove unbound staples (see: 2011/09/06), its concentration can only be roughly estimated to 10 nM.

This solution is diluted as follows in FOB20:

  • 1:100
  • 1:200
  • 1:500
  • 1:1000
  • 1:10000

Fluorescence microscope setup

  • Exposure Time: 50 ms
  • Series: Videos of 50 frames
  • Alternating excitation with green/red laser, simultanous detection on green and red camera

Results

In the 1:100 dilution, the space is densely crowded with origamis. The 1:10000 dilution shows a density, at which single structures can be picked conviniently for single molecule analysis.
FRET events could be observed, i.e. spots at which the Atto 550 fluorescence intensity increased after photobleaching of Atto 647N.


Figure 1: Structure BM_5/20, green laser, dilution 1:100
Figure 2: Structure BM_5/20, green laser, dilution 1:10000