Biomod/2011/TUM/TNT/LabbookA/2011/08/24: Difference between revisions

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<h6><b>13<font size="-1">th</font> Sep 2011</b></h6>
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<div id="struktur">
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<center><h1>'''24<font size="-1">th</font> Aug 2011'''</h1></center>
 
<h2> Purification of the U structure from 15_65 thermal ramp by agarose gel electrophoresis</h2>
<h2> Purification of the U structure from 15_65 thermal ramp by agarose gel electrophoresis</h2>


Revision as of 17:23, 29 October 2011

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       TUM NanU - Home

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   <li><a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/Extras">Extras</a>
       
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           <a href="http://openwetware.org/wiki/Biomod/2011/TUM/TNT/LabbookA/2011/TIRF">Fluorescence microscopy</a>|
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Purification of the U structure from 15_65 thermal ramp by agarose gel electrophoresis


Figure 1: From left: 1kb-ladder, scaffold p7560, BM1, BM2


The gel displays a major band for BM1 and BM2 as well as other quite weak bands (probably byproducts).

The strong bands were cut out and thoroughly shredded with mortar and pestle. The agarose debris is spun down. Then the tip of the Eppendorf tube, which contains the agarose debris, is cut off using a razor blade. The inverted tip is put into a Freeze 'N Squeeze DNA Gel Extraction Spin Column. After 10 minutes centrifuging at maximum speed, the origami structure is eluted and can be used for further examination.

Analysis with the TEM

Sample preparation according to standard protocol.

Labeling oligos with fluorophores

Labeling of the oligos was done by using terminal transferase according to standard protocol.
All reactions were accomplished twice, but for the staples "the_U 218" and "the_U 225" only one reaction was successful.
Labeled oligos:

  • the_U 175 with Atto550_ddCTP helix 4
  • the_U 218 with Atto647N_ddUTP helix 23


  • the_U 197 with Atto550_ddCTP helix 8
  • the_U 225 with Atto647N_ddUTP helix 29


  • the_U 183 with Atto550_ddCTP helix 5
  • the_U 204 with Atto647N_ddUTP helix 20


For purification, batches of identical probes were combined.
Labeled oligos were resuspended in 100 µl buffer (0.5xTBE 11mM MgCl2); 50µl for the_U 218 and the_U 255 each.

Preparation of solutions for determining absorption coefficients of unlabeled oligonucleotides according to standard protocol


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