Biomod/2013/Todai/Experiment

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
Line 19: Line 19:
.mini-title a {
.mini-title a {
   font-size :130%;
   font-size :130%;
 +
  font-weight:bolder;
   display: block;
   display: block;
   text-decoration: none;
   text-decoration: none;
Line 25: Line 26:
   left:25px;
   left:25px;
   line-height: 1.5;
   line-height: 1.5;
-
  border-bottom: solid 2px black;
 
   margin-bottom: 10px;
   margin-bottom: 10px;
   }
   }
Line 82: Line 82:
   }
   }
-
h2 a{
+
.PS_title a{
   color:black;
   color:black;
   text-decoration:none;
   text-decoration:none;
Line 97: Line 97:
<body>
<body>
-
<!--◆◆Experiment◆◆-->
+
<!--Experiment-->
     <h1 class="big-title"><a name="Experiment">&nbsp;Experiment</a></h1>
     <h1 class="big-title"><a name="Experiment">&nbsp;Experiment</a></h1>
     <div id="Explist">
     <div id="Explist">
     <ul >
     <ul >
-
 
       <li><div class="mokuji"><a href="#Contents">Contents</a></div></li>
       <li><div class="mokuji"><a href="#Contents">Contents</a></div></li>
       <li><div class="mokuji"><a href="#PilotStudy">Pilot Study</a></div></li>
       <li><div class="mokuji"><a href="#PilotStudy">Pilot Study</a></div></li>
Line 109: Line 108:
     <br>
     <br>
-
<!--◆◆Contents◆◆-->
+
<!--Contents-->
   <article>
   <article>
     <h1 class="title"><a name="Contents">&nbsp;Contents</a></h1>
     <h1 class="title"><a name="Contents">&nbsp;Contents</a></h1>
-
  <article>
+
      <article>
-
    <ul class="Contents-list">
+
        <ul class="Contents-list">
-
 
+
-
  <li><a name="#Assembling_of_DNA_structure"><b>Assembly of DNA nanostructure</b></a> -->(See <a href="http://openwetware.org/wiki/Biomod/2013/Todai/Result">Result</a>)
+
        <li>
-
<div class="preparation">Optimize the assembly condition of the DNA nanostructure,"Cylinder in barrel"</div><br>
+
        <a name="#Assembling_of_DNA_structure">
-
</li>
+
        <b>Assembly of DNA nanostructure</b>
-
<li><a name="#flotation_assay"><b>Flotation assay</b></a>
+
        </a>
-
    <ul class="Contents-list">
+
        -->(See <a href="http://openwetware.org/wiki/Biomod/2013/Todai/Result" style="color:#E00000">Result</a>)
-
      <li><a href ="#Preparation_of_liposome">Preparation of liposome</a><br>
+
        <br>
-
      Making of liposome for floatation assay
+
        Optimize the assembly condition of the DNA nanostructure,"Cylinder in barrel"
-
      <br>
+
        </li>
-
      </li>
+
        <br>
-
      <li><a href ="#Flotation_assay_of_liposome_and_DNA_origami">Floatation assay of liposome and DNA origami</a>
+
-
<br> Assay to check the penetration of DNA origami
+
-
<br>
+
-
</li>
+
-
</ul><br></li>
+
-
<li>
+
-
<a href ="#Comparision_of_dimerization"><b>Comparision of dimerization method</b> </a>
+
-
<ul class="Contents-list">
 
-
      <li><a href ="#Click_reaction_via_(3+2)_cycloaddition">Click reaction via (3+2) cycloaddition</a>
 
-
<br>Optimize the reaction condition for click chemistry
 
-
<br>
 
-
</li>
 
-
<li><a href ="#Azobenzene">Azobenzene</a>
 
-
<ul class="Contents-list">
+
        <li><a name="#flotation_assay"><b>Flotation assay</b></a>
-
      <li><a href ="#Synthesis_of_Tube(Research_for_azobenzene)">Synthesis of tube</a>
+
        <ul class="Contents-list">
-
<br> Optimize the assembly condition pf the DNA origami tube to be equipped with azobenzene
+
          <li><a href ="#Preparation_of_liposome">Preparation of liposome</a>
-
<br>
+
          <br>
-
</li>
+
          Making of liposome for floatation assay
 +
        </li>
 +
   
 +
        <li>
 +
        <a href ="#Flotation_assay_of_liposome_and_DNA_origami">
 +
        Floatation assay of liposome and DNA origami
 +
        </a>
 +
<br>
 +
        Assay to check the penetration of DNA origami
 +
<br>
 +
</li>
 +
        </ul>
 +
        <br>
-
<li><a href ="#Synthesis_of_Motif(Research_for_azobenzene)">Synthesis of tube</a>
+
        <li>
-
<br>Optimize the assembly condition of the DNA motif to be equipped with azobenzene<br>
+
        <a href ="#Comparision_of_dimerization">
-
</li>
+
        <b>Comparision of dimerization method</b>
-
</ul>
+
        </a>
-
</li>
+
        <ul class="Contents-list">
 +
          <li>
 +
          <a href ="#Click_reaction_via_(3+2)_cycloaddition">
 +
          Click reaction via (3+2) cycloaddition
 +
          </a>
 +
          <br>
 +
          Optimize the reaction condition for click chemistry
 +
          <br>
 +
          </li>
-
</ul>
+
          <li><a href ="#Azobenzene">Azobenzene</a>
-
</li>
+
          <ul class="Contents-list">
-
        
+
            <li><a href ="#Synthesis_of_Tube(Research_for_azobenzene)">Synthesis of tube</a>
-
    </ul>
+
            <br>
-
</article>
+
            Optimize the assembly condition pf the DNA origami tube to be equipped with azobenzene
-
</article>
+
            <br>
-
<!--◆◆Pilot Study◆◆-->
+
            </li>
 +
 
 +
            <li><a href ="#Synthesis_of_Motif(Research_for_azobenzene)">Synthesis of tube</a>
 +
            <br>
 +
            Optimize the assembly condition of the DNA motif to be equipped with azobenzene
 +
            </li>
 +
            <br>
 +
 
 +
          </ul>
 +
 
 +
          </li>
 +
 
 +
       </ul>
 +
      </li>
 +
      </ul>
 +
  </article>
 +
</article>
 +
 
 +
 
 +
<!--Pilot Study-->
   <article>
   <article>
Line 168: Line 192:
     <h1 class="title"><a name="PilotStudy">&nbsp;Pilot Study</a></h1>
     <h1 class="title"><a name="PilotStudy">&nbsp;Pilot Study</a></h1>
      
      
-
<!--★Liposome making★-->
+
<!--Liposome making-->
   <article>
   <article>
    
    
-
   <h2><a name="Assembling_of_DNA_structure">&nbsp;1.Assembly of DNA nanostructure</a></h2>
+
   <h2 class="PS_title"><a name="Assembling_of_DNA_structure">&nbsp;1.Assembly of DNA nanostructure</a></h2>
   <p class="paragraph">
   <p class="paragraph">
-
   The assembly of DNA structure is explained in <a href="http://openwetware.org/wiki/Biomod/2013/Todai/Result">result</a>.
+
   The assembly of DNA structure is explained in the
-
   <h2><a name="flotation_assay">&nbsp;2.Flotation assay</a></h2>
+
  <a href="http://openwetware.org/wiki/Biomod/2013/Todai/Result" style="color:#e00000">Result page</a>.
 +
  <br> 
 +
  <br>
 +
 
 +
   <h2 class="PS_title"><a name="flotation_assay">&nbsp;2.Flotation assay</a></h2>
   <div class="mini-title">
   <div class="mini-title">
-
    <div style="width:512px">
+
       <a name="Preparation_of_liposome">2-1. Preparation of liposome</a>
-
       <a name="Preparation_of_liposome">2.1.Preparation of liposome</a>
+
-
    </div>
+
   </div>
   </div>
Line 199: Line 225:
   <br>
   <br>
-
<!--★Flotation assay★-->
+
<!--Flotation assay-->
 +
 
   <article>
   <article>
   <div class="mini-title">
   <div class="mini-title">
-
    <div style="width:512px">
+
       <a name="Flotation_assay_of_liposome_and_DNA_origami">2-2. Floatation assay of liposome and DNA origami</a>
-
       <a name="Flotation_assay_of_liposome_and_DNA_origami">2.2.Floatation assay of liposome and DNA origami</a>
+
-
  </div>
+
       <figure>
       <figure>
         <center>
         <center>
Line 233: Line 258:
   <br>
   <br>
</article>
</article>
-
<!--★Click Reaction★-->
+
 
-
<h2><a name="Comparision_of_dimerization">3.Comparision of dimerization method</a></h2>
+
 
 +
<!--Click Reaction-->
 +
  <h2 class="PS_title"><a name="Comparision_of_dimerization">3. Comparision of dimerization method</a></h2>
   <article>
   <article>
   <div class="mini-title">
   <div class="mini-title">
-
    <div style="width:500px">
 
       <a name="Click_reaction_via_(3+2)_cycloaddition">
       <a name="Click_reaction_via_(3+2)_cycloaddition">
-
       3-1.Click reaction via (3+2) cycloaddition<sup>[4]</sup>
+
       3-1. Click reaction via (3+2) cycloaddition<sup>[4]</sup>
       </a>
       </a>
-
    </div>
 
   </div>
   </div>
       <figure>
       <figure>
Line 259: Line 284:
   <br>
   <br>
-
<!--★Synthesis of tube (Research for azobenzene)-->
+
<!--Synthesis of tube (Research for azobenzene)-->
   <article>
   <article>
       <div class="mini-title">
       <div class="mini-title">
-
    <div style="width:150px">
+
       <a name ="Azobenzene">3-2. Azobenzene</a>
-
       <a name ="Azobenzene">3-2.Azobenzene</a>
+
     </div>
     </div>
-
  </div>
+
 
-
  <div class="mini-title">
+
    <div class="mini-title">
-
    <div style="width:512px">
+
       <a name="Synthesis_of_Tube(Research_for_azobenzene)">&nbsp;&nbsp;3-2-1. Synthesis of tube<sup>[2],[3]</sup>(Research for azobenzene)</a>
-
       <a name="Synthesis_of_Tube(Research_for_azobenzene)">3-2-1.Synthesis of tube<sup>[2],[3]</sup>(Research for  
+
-
azobenzene)</a>
+
     </div>
     </div>
-
  </div>
 
       <figure>
       <figure>
         <center>
         <center>
         <img src="http://openwetware.org/images/b/b2/640x360px_tube_result-Todai.png" width=640px height=360px >
         <img src="http://openwetware.org/images/b/b2/640x360px_tube_result-Todai.png" width=640px height=360px >
-
<figcaption> <b>results of the electrophoresis of DNA-tube</b><br>
+
        <figcaption>
-
 
+
        <b>results of the electrophoresis of DNA-tube</b><br>
-
      </figcaption>
+
        </figcaption>
         </center>
         </center>
       </figure>
       </figure>
-
<div class="zairyou-heading">[Discussion]</div>
+
 
 +
    <div class="zairyou-heading">[Discussion]</div>
     <p class="paragraph">  
     <p class="paragraph">  
-
We examined how DNA-tube was synthesized efficiently by using the method which is introduced in “Rapid Folding of  
+
    We examined how DNA-tube was synthesized efficiently by using the method which is introduced in “Rapid Folding of  
-
 
+
DNA into Nanoscale Shapes at Constant Temperature” (Jean-Philippe J. Sobczak et al, Science, 2012, 338, 1458)
DNA into Nanoscale Shapes at Constant Temperature” (Jean-Philippe J. Sobczak et al, Science, 2012, 338, 1458)
-
 
+
    <sup>[2]</sup>. In the figure above, two bands derived from scaffold or DNA-tube were showed with cursors. At 56.4℃, the scaffold  
-
<sup>[2]</sup>. In  
+
-
 
+
-
the figure above, two bands derived from scaffold or DNA-tube were showed with cursors. At 56.4℃, the scaffold  
+
-
 
+
band was diminished. In contrast, the DNA-tube band was concentrated. In fact, the ratio of DNA- tube band  
band was diminished. In contrast, the DNA-tube band was concentrated. In fact, the ratio of DNA- tube band  
-
 
to scaffold band was the greatest at this temperature, which means we succeeded in synthesizing DNA-
to scaffold band was the greatest at this temperature, which means we succeeded in synthesizing DNA-
-
 
tube efficiently and improving the yield of DNA-tube.
tube efficiently and improving the yield of DNA-tube.
-
 
     </p>
     </p>
   <br>
   <br>
-
<!--★Synthesis of Motif (Research for azobenzene)-->
+
 
 +
 
 +
<!--Synthesis of Motif (Research for azobenzene)-->
   <article>
   <article>
   <div class="mini-title">
   <div class="mini-title">
-
    <div style="width:256px">
+
       <a name="Synthesis_of_Motif(Research_for_azobenzene)">&nbsp;&nbsp;3-2-2. Synthesis of Motif<sup>[5]</sup></a>
-
       <a name="Synthesis_of_Motif(Research_for_azobenzene)">3.2.2.Synthesis of Motif<sup>[5]</sup></a>
+
-
    </div>
+
   </div>
   </div>
       <figure>
       <figure>
         <center>
         <center>
-
         <img src="http://openwetware.org/images/a/a8/480px_electrophoresis_of_T-motif_improved-Todai.png"  
+
         <img src="http://openwetware.org/images/a/a8/480px_electrophoresis_of_T-motif_improved-Todai.png" width=480px height=360px >
-
 
+
        <figcaption>
-
width=480px height=360px >
+
          <b>Result of agarose gel electrophoresis of T-motif (wheel)</b>
-
<figcaption> <b>Result of agarose gel electrophoresis of T-motif (wheel)</b><br>
+
          <br>
-
The result of 1.5% agarose gel electrophoresis(100V) for 33min.  
+
          The result of 1.5% agarose gel electrophoresis(100V) for 33min.  
-
      </figcaption>
+
        </figcaption>
         </center>
         </center>
       </figure>
       </figure>
-
<div class="zairyou-heading">[Discussion]</div>
+
 
 +
    <div class="zairyou-heading">[Discussion]</div>
     <p class="paragraph">  
     <p class="paragraph">  
-
In this measurement, it is difficult to distinguish between final structure band and monomer band.  
+
    In this measurement, it is difficult to distinguish between final structure band and monomer band.
-
Next time, Native PAGE will be used instead of agarose gel.  
+
    Next time, Native PAGE will be used instead of agarose gel.  
     </p>
     </p>
   <br>
   <br>
-
<!--◆◆Protocols◆◆-->
+
<!--Protocols-->
   <article>
   <article>
     <h1 class="title"><a name="Protocols">&nbsp;Protocols</a></h1>
     <h1 class="title"><a name="Protocols">&nbsp;Protocols</a></h1>
-
<!--★定温法 構造体(staple+コレステ)組立プロトコル★-->
+
<!--scaffold+staple+cholesterol-->
   <article>
   <article>
   <div class="mini-title">
   <div class="mini-title">
-
    <div style="width:515px">
+
       <a name="Assembling_of_DNA structure (scaffold+staple+cholesterol)">1.Assembly of DNA structure (scaffold+staple)<sup>[2]</sup></a>
-
       <a name="Assembling_of_DNA structure (scaffold+staple+cholesterol)">1.Assembly of DNA structure (scaffold
+
-
 
+
-
+staple)<sup>[2]</sup></a>
+
-
    </div>
+
   </div>
   </div>
Line 373: Line 384:
     <br>
     <br>
-
<!--★リポソーム作製★-->
+
<!--liposome-->
   <article>
   <article>
   <h2>2.Flotation assay</h2>
   <h2>2.Flotation assay</h2>
   <div class="mini-title">
   <div class="mini-title">
-
    <div style="width:215px">
+
       <a name="Preparation_of_liposome">2-1. Preparation of liposome</a>
-
       <a name="Preparation_of_liposome">2.1.Preparation of liposome</a>
+
-
    </div>
+
   </div>
   </div>
Line 427: Line 436:
     <br>
     <br>
-
<!--★フローテーションアッセイ★-->
+
<!--flotation assay-->
   <article>
   <article>
   <div class="mini-title">
   <div class="mini-title">
-
    <div style="width:512px">
+
       <a name="Flotation_assay_of_liposome">2-2. Flotation assay of liposome and DNA origami</a>
-
       <a name="Flotation_assay_of_liposome">2.2.Flotation assay of liposome and DNA origami</a>
+
-
    </div>
+
   </div>
   </div>
Line 511: Line 518:
     </table>
     </table>
     <br>
     <br>
-
     <div class="zairyou-heading-sub">・liposome </div>    <div style="font-
+
     <div class="zairyou-heading-sub">・liposome </div>
-
 
+
     <div style="font-size:110%;position:relative;left:60px;">
-
size:110%;position:relative;left:60px;">
+
    1mg/ml( -->
-
1mg/ml( -->
+
    <a href="#Preparation_of_liposome" style="color:#e00000">Preparation of liposome</a>)
-
        <a href="#Preparation_of_liposome" style="color:#e00000">Preparation of liposome</a>)
+
    </div>
-
    </div>
+
     <div class="zairyou-heading-sub">・DNA origami(Rect tile<sup>[1]</sup>) </div>
     <div class="zairyou-heading-sub">・DNA origami(Rect tile<sup>[1]</sup>) </div>
-
    <div style="font-size:110%;position:relative;left:60px;">~30nM
+
    <div style="font-size:110%;position:relative;left:60px;">~30nM</div>
-
    </div>
+
-
 
+
-
 
+
     <br>
     <br>
Line 531: Line 534:
   <div class="zairyou-heading">[Procedure]</div>
   <div class="zairyou-heading">[Procedure]</div>
     <ul class="procedure-list">
     <ul class="procedure-list">
-
       <li>sample + sucrose buffer were layered  
+
       <li>sample + sucrose buffer were layered in this order on centrifuge tubes.</li>
-
in this order on centrifuge tubes.</li>
+
       <li>The tubes were centrifugalized.</li>
       <li>The tubes were centrifugalized.</li>
       <li>Samples were fructionized.</li>
       <li>Samples were fructionized.</li>
-
       <li>Precipitation was resuspended with sucrose buffer.
+
       <li>Precipitation was resuspended with sucrose buffer.</li>
-
       <li>Samples were measured by the fluorescence of NIL(ex 500nm, em 550nm~700nm) and agarose gel  
+
       <li>Samples were measured by the fluorescence of NIL(ex 500nm, em 550nm~700nm) and agarose gel electrophoresis.</li>
-
 
+
-
electrophoresis.</li>
+
     </ul>
     </ul>
     </article>
     </article>
Line 544: Line 544:
-
 
+
<!--Click Reaction-->
-
<!--★Click Reaction★-->
+
   <article>
   <article>
-
   <h2>3.Comparision of dimerization method</h2>
+
   <h2>3. Comparision of dimerization method</h2>
   <div class="mini-title">
   <div class="mini-title">
-
    <div style="width:350px">
 
       <a name="Click_reaction_via_(3+2)_cycloaddition">
       <a name="Click_reaction_via_(3+2)_cycloaddition">
-
       3.1.Click reaction via (3+2) cycloaddition
+
       3-1. Click reaction via (3+2) cycloaddition
       </a>
       </a>
-
    </div>
 
   </div>
   </div>
Line 598: Line 595:
     </article>
     </article>
     <br>
     <br>
-
<!--★Research for azobenzene★-->
+
 
 +
<!--Research for azobenzene-->
 +
 
   <article>
   <article>
   <div class="mini-title">
   <div class="mini-title">
-
    <div style="width:350px">
 
       <a name="Research_for_azobenzene)">
       <a name="Research_for_azobenzene)">
-
       3.2.Research for azobenzene
+
       3-2. Research for azobenzene
       </a>
       </a>
-
    </div>
 
   </div>
   </div>
Line 611: Line 608:
<!--Procedure-->
<!--Procedure-->
   <div class="zairyou-heading">[Procedure]<sup>[2],[3],[5]</sup></div>
   <div class="zairyou-heading">[Procedure]<sup>[2],[3],[5]</sup></div>
-
  <p class="paragraph">
+
    <p class="paragraph">
-
The procedure of synthesis of tubes and motifs were refered to previous reseachs([2],[3],[5])  
+
    The procedure of synthesis of tubes and motifs were refered to previous researches([2],[3],[5])  
-
 
+
    </p>
-
 
+
-
 
+
     </article>
     </article>
     <br>
     <br>
-
  <div class="mini-title">
+
    <br>
-
    <div style="width:200px">
+
 
-
      <a name="Reference">
+
-
      Reference
+
-
      </a>
+
-
    </div>
+
-
  </div>
+
 +
<!--Reference-->
 +
    <h1 class="title"><a name="Reference">&nbsp;Reference</a></h1>
     <div>     
     <div>     
Line 640: Line 632:
               Nature 440, 297–302 (2006)
               Nature 440, 297–302 (2006)
               </div>
               </div>
-
     <div>    
+
    </div>
 +
 
 +
     <div>
         <div class="reference-title">
         <div class="reference-title">
         <a name="proref-1">
         <a name="proref-1">
Line 652: Line 646:
               Science, 2012, 338, 1458
               Science, 2012, 338, 1458
               </div>
               </div>
 +
    </div>
 +
     <div>     
     <div>     
         <div class="reference-title">
         <div class="reference-title">
         <a name="proref-1">
         <a name="proref-1">
-
         [3] Transcription Regulation System Mediated by Mechanical Operation of a DNA Nanostructure
+
         [3] Transcription Regulation System Mediated by Mechanical Operation of a DNA &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Nanostructure
         </a>
         </a>
         </div>
         </div>
           <div class="reference-author">
           <div class="reference-author">
-
           >Masayuki Endo, Ryoji Miyazaki, Tomoko Emura, Kumi Hidaka, and Hiroshi Sugiyama
+
           Masayuki Endo, Ryoji Miyazaki, Tomoko Emura, Kumi Hidaka, and Hiroshi Sugiyama
           </div>
           </div>
               <div class="reference-journal">
               <div class="reference-journal">
               Journal of the American Chemical Society, 2012, 134, 2852-2855
               Journal of the American Chemical Society, 2012, 134, 2852-2855
               </div>
               </div>
 +
    </div>
 +
     <div>     
     <div>     
         <div class="reference-title">
         <div class="reference-title">
Line 670: Line 668:
         </a>
         </a>
         </div>
         </div>
-
              <div class="reference-journal">
+
          <div class="reference-journal">
-
             
+
          <a target="_blank" href="http://www.jenabioscience.com" style="color:#e00000">
-
      <a target="_blank" href="http://www.jenabioscience.com" style="color:#e00000">
+
          http://www.jenabioscience.com</a>
-
      http://www.jenabioscience.com</a>
+
          </div>
-
              </div>
+
    </div>
-
     <div>    
+
 
 +
     <div>
         <div class="reference-title">
         <div class="reference-title">
         <a name="proref-1">
         <a name="proref-1">
Line 687: Line 686:
               Angewandte Chemie International Edition,2009,48(37),6820–6823
               Angewandte Chemie International Edition,2009,48(37),6820–6823
               </div>
               </div>
 +
    </div>
 +
                
                
   </article>
   </article>

Revision as of 23:57, 18 September 2013


Experiment-Todai nanORFEVRE-

 Experiment


 Contents

 Pilot Study


2-2. Floatation assay of liposome and DNA origami
Result of agarose gel electrophoresis of the sample of flotation assay
The result of 1% agarose gel electrophoresis(100V,30min). In this measurement, the fluorescence of Cy5, which is integrated into DNA origami(Rect tile[1]) ,is observed. Fraction1 is the liquid in the top layer, and fraction 5 is in the bottom layer. Fraction 6 is the sample retrieved from precipitation. DNA origami solely was also loaded on the extreme right lane.
Fluorescence intensity of the samples of flotation assay(DNA Rect tile +liposome)
Although the size of liposome might change during the flotation assay(data not shown), the intensity of the fluorescence of NIL(Nile Red, ex 500nm, em 550~700nm ) suggests the amount of lipid membrane,liposome. The fluorescence spectrum of water was subtracted as background

[Discussion]

To confirm the flotation assay, mixed tiles(DNA origami) and liposomes were assayed. Five samples (fraction 1,2,...,5, from the top) were retrieved from supermetant liquid and a sample(fraction 6) from precipitation by the addition of buffer used in assay. When the sample, tile mixed with liposomes, were assayed, tiles were observed in the top layer. The distribution of liposomes is observed by the fluorescence of NIL(Nile Red).


3. Comparision of dimerization method

Result of urea gel electrophoresis of the sample of click reaction
[Discussion]

Copper(Ⅰ) catalyzed click reaction was used to dimerize of oligo DNA(length of 20bp and 14bp). The time cause of the reaction indicate that the click reaction is so quick(<5min).


results of the electrophoresis of DNA-tube
[Discussion]

We examined how DNA-tube was synthesized efficiently by using the method which is introduced in “Rapid Folding of DNA into Nanoscale Shapes at Constant Temperature” (Jean-Philippe J. Sobczak et al, Science, 2012, 338, 1458) [2]. In the figure above, two bands derived from scaffold or DNA-tube were showed with cursors. At 56.4℃, the scaffold band was diminished. In contrast, the DNA-tube band was concentrated. In fact, the ratio of DNA- tube band to scaffold band was the greatest at this temperature, which means we succeeded in synthesizing DNA- tube efficiently and improving the yield of DNA-tube.


Result of agarose gel electrophoresis of T-motif (wheel)
The result of 1.5% agarose gel electrophoresis(100V) for 33min.
[Discussion]

In this measurement, it is difficult to distinguish between final structure band and monomer band. Next time, Native PAGE will be used instead of agarose gel.


 Protocols

[Reagent]

M13mp18ss (0.1μM) 5μL
staple mix (0.68μM) 4μL
10x buffer 1μL

[Procedure][2]
  • 5μL of M13mp18ss, 4μL of staple mix and 1μL of 10×buffer were mixed, pipetting.
  • The solution was kept at 45 degree C for 4h.

2.Flotation assay

[Reagent]

150mM aqueous KCl solution 3mL
POPC 3mg
Chloroform (99.0%) 3mL
40μM Nile Red solution 0.1mL

[Procedure]
  • POPC were dissolved in 3mL of Chloroform.
  • A lipid film was formed by evaporating 3mL of POPC solution in a 50mL eggplant flask, using a rotational evaporator for 5 minutes.
  • The flask was kept under vacuum overnight to evaporate remaining chloroform.
  • The lipid film was resuspended in 3mL of a 150mM aqueous KCl solution.
  • The solution was filtered through 200nm polar filter with extruder to even the size of liposome.
  • The size of liposome was measured with DLS (Viscotek 802 DLS).
  • The solution was kept at 3 degree C until usage.

[Reagent]
・Sucrosebuffer
a) 0.375M Sucrose buffer
HEPES-KOH(pH7.6) 50mM
KCl 100mM
MgCl2 10mM
Sucrose 0.375M
b) 1.25M Sucrose buffer
HEPES-KOH(pH7.6) 50mM
KCl 100mM
MgCl2 10mM
Sucrose 1.25M
c) 1.6M Sucrose buffer
HEPES-KOH(pH7.6) 50mM
KCl 100mM
MgCl2 10mM
Sucrose 1.6M

・liposome
・DNA origami(Rect tile[1])
~30nM

[Procedure]
  • sample + sucrose buffer were layered in this order on centrifuge tubes.
  • The tubes were centrifugalized.
  • Samples were fructionized.
  • Precipitation was resuspended with sucrose buffer.
  • Samples were measured by the fluorescence of NIL(ex 500nm, em 550nm~700nm) and agarose gel electrophoresis.

3. Comparision of dimerization method

[Reagent]

azide solution (10μM) 3μL
alkyne solution (10μM) 3μL
CuSO4 solution (50mM) 1μL
THTA solution (100mM) 1μL
sodium ascorbate solution (100mM) 1μL

[Procedure][4]
  • The above all solutions were mixed, using a vortex.
  • The solution was kept at room temperature.



 Reference

Rothemund, P. W.
Nature 440, 297–302 (2006)
Jean-Philippe J. Sobczak, Thomas G. Martin, Thomas Gerling, Hendrik Dietz
Science, 2012, 338, 1458
Masayuki Endo, Ryoji Miyazaki, Tomoko Emura, Kumi Hidaka, and Hiroshi Sugiyama
Journal of the American Chemical Society, 2012, 134, 2852-2855
Shogo Hamada, Satoshi Murata Prof.
Angewandte Chemie International Edition,2009,48(37),6820–6823


Personal tools