Biomod/2011/TUM/TNT/LabbookA/2011/10/19

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19th Oct 2011 <!-- sibboleth --><div id="m1" style="border:0px;"><div style="display:none;" id="id">m1</div><div style="display:none;" id="dtext"></div><div style="display:none;" id="page">Biomod/2011/TUM/TNT/LabbookA/20</div><div style="display:none;" id="fmt">yyyy/MM/dd</div><div style="display:none;" id="css">OWWNB</div><div style="display:none;" id="month">8</div><div style="display:none;" id="year">2011</div><div style="display:none;" id="readonly">Y</div></div> <!-- sibboleth --><div id="m2" style="border:0px;"><div style="display:none;" id="id">m2</div><div style="display:none;" id="dtext"></div><div style="display:none;" id="page">Biomod/2011/TUM/TNT/LabbookA/20</div><div style="display:none;" id="fmt">yyyy/MM/dd</div><div style="display:none;" id="css">OWWNB</div><div style="display:none;" id="month">9</div><div style="display:none;" id="year">2011</div><div style="display:none;" id="readonly">Y</div></div> <!-- sibboleth --><div id="m3" style="border:0px;"><div style="display:none;" id="id">m3</div><div style="display:none;" id="dtext"></div><div style="display:none;" id="page">Biomod/2011/TUM/TNT/LabbookA/20</div><div style="display:none;" id="fmt">yyyy/MM/dd</div><div style="display:none;" id="css">OWWNB</div><div style="display:none;" id="month">10</div><div style="display:none;" id="year">2011</div><div style="display:none;" id="readonly">Y</div></div>

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TIRF

Camera Calibration

In order to make quantitative measurements of the distance of two flourophores we had to calibrate our fluorescence microscope. Therefore we took a picture of 5 scale bars (10 µm middle to middle).
Hence the orientation isn't exactly vertically, we measured the horizontal shift of the bars to get the angle. With this we can calculate the right distance in pixels between the bars. So our calibration is:
Failed to parse (MathML with SVG or PNG fallback (recommended for modern browsers and accessibility tools): Invalid response ("Math extension cannot connect to Restbase.") from server "https://api.formulasearchengine.com/v1/":): {\displaystyle 101.03 nm/px }

Calibration file: File:Calibration cam green.xls

Structure

Determined Concentrations of labeled Oligonucleotides

  • Staple 110 (helix 4) with ddUTP Atto 550: 0.7 µM
  • Staple 164 (helix 29) with ddUTP Atto 647N: 3.2 µM

Folded Structure BM25_4/29

BM25_4/29 is intrinsically twisted, as BM24, but with fluorophors at staple 110 (helix 4, ddUTP Atto 550) and staple 164 (helix 29, dd UTP Atto 647N).
10 µl each of both labeled oligonucleotides were added to the folding batch, replacing the usual 20µl ddH2O. Folding ramps 15_65 and 2D_H3_ML were applied for one folding batch each.