Biomod/2014/Kashiwa/Protocol: Difference between revisions
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Revision as of 02:41, 21 August 2014
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<ul id="menu"> <font face="Frutiger,Helvetica,Arial"> <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa"><img src="http://openwetware.org/images/e/ec/LogoKashiwa.png" onmouseover="this.src='http://openwetware.org/images/7/7a/Logo2Kashiwa.png'" onclick="this.src='http://openwetware.org/images/1/1d/Logo2.5.png'" onmouseout="this.src='http://openwetware.org/images/e/ec/LogoKashiwa.png'" height="80px" width="120px" name="def"></a> </li> <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Project" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">PROJECT</span></a> <ul class="sub"> <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Project#1" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Background</span></a></li> <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Project#2" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Motivation</span></a></li> <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Project#3" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Project Goals</span></a></li> </ul> </li> <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Trial" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;"> EARLY TRIAL </span></a> <ul class="sub"> <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Trial#1" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Design</span></a></li> <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Trial#2" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Approaches</span></a></li> </ul> </li> <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Design" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">DESIGN</span></a> <ul class="sub"> <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Design#2" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">The Receptor</span></a></li> <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Design#1" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">The Motor</span></a></li> </ul> </li> <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Highlights" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;"> EXPERIMENT </span></a> <ul class="sub"> <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Highlights" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Highlights</span></a></li> <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Receptor" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">The Receptor</span></a></li> <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Motor" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">The Motor</span></a></li> </ul> <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Discussion" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;"> DISCUSSION </span></a> <ul class="sub"> <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Discussion#1" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Achievements</span></a></li> <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Discussion#2" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Future</span></a></li> </ul> </li> <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Protocols" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">PROTOCOL</span></a> </li> <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Team" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">TEAM</span></a> <ul class="sub"> <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Team#1" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Members</span></a></li> <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Team#2" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">Sponsors</span></a></li> </ul> </li> </font> </ul>
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<body> <font face="Futura,Arial,Frutiger" font size="24pt">PROTOCOL</font> <br> <br> <div class="CollapsibleBox" id="CBoxCover1">
<div class="CollapsibleBoxTitle"> <p class="cBoxButtons"> <a href="#" onclick="HideCBox('CBoxBody1'); return false;" title="折りたたみ/復元"><font color="white" font size="2">[show/hide]</font></a> </p> <p class="cBoxTitle"> Contents:</p> </div> <div class="CollapsibleBoxBody" id="CBoxBody1"> <ul style="list-style:none;"> <li> <a href=#1>1. Polymerization </a> <ul style="list-style:none;"> <li> <a href="#1-1">1-1. Making the Monomer </a></li> <li> <a href="#1-2">1-2. Controlling polymerization </a></li> <li> <a href="#1-3">1-3. Putting the Monomer into a liposome </a></li> <li> <a href="#1-4">1-4. Polymerization </a> <ul style="list-style:none;"> <li> <a href="#1-4-1">1-4-1. Polymerization in solution </a></li> <li> <a href="#1-4-2">1-4-2. Polymerization in a liposome </a></li> </ul> </li> <li> <a href="#1-5">1-5. Deformation of a liposome </a></li> </ul> </li> <li> <a href="#2">2. Receptor </a> <ul style="list-style:none"> <li> <a href="#2-1">2-1. Making the Receptor </a></li> <li> <a href="#2-2">2-2. Penetration into a liposome </a></li> <li> <a href="#2-3">2-3. Dimerization <ul style="list-style:none"> <li> <a href="#2-3-1">2-3-1. Dimerization in solution </a></li> <li> <a href="#2-3-2">2-3-2. Dimerization on a liposome </a></li> </ul> </li> <li> <a name="1"><a href="#2-4">2-4. Emission of polymerization initiator by dimerization </a></a> <ul style="list-style:none"> <li> <a href="#2-4-1">2-4-1. Emission by dimerization in solution </a></li> <li> <a name="1-1"><a href="#2-4-2">2-4-2. Emission by dimerization on a liposome </a></a></li> </ul> </li> </ul> </ul> </div>
</div> <h1 class="title"><a name="contents"> 1. Polymerization</a></h1>
<p class="small">1-1. Making the monomer</p>
<table class="sample_01">
<tbody>
<tr>
<th>Reagents (f.60 µL)</th>
<th> </th>
</tr>
<tr>
<td>Staple mix</td>
<td>17.5 µL</td>
</tr>
<tr>
<td>M13mp18ss</td>
<td>18.3 µL</td>
</tr>
<tr>
<td>TE¹</td>
<td>18.2 µL</td>
<tr>
<td>10 × tile buffer²</td>
<td>6.0 µL</td>
</tr>
</tr>
</tbody>
</table>
<table class="sample_02">
<tbody>
<tr>
<th>¹TE(f.50 mL)</th>
<th> </th>
<th> </th>
</tr>
<tr>
<td>1M Tris-HCl(pH8.0)</td>
<td>0.5 mL</td>
<td>(f.10 mM)
</tr>
<tr>
<td>0.5M EDTA</td>
<td>0.1 mL</td>
<td>(f.1 mM)</td>
</tr>
</tr>
</tbody>
</table>
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<tbody>
<tr>
<th>²10 × tile buffer(f.500 mL)</th>
<th> </th>
<th> </th>
</tr>
<tr>
<td>Mg(OAc)<sub>2</sub></td>
<td>10.73 g</td>
<td>f.100 mM
</tr>
<tr>
<td>1.0M Tris-HCl(pH7.5)</td>
<td>100 mL</td>
<td>f.200 mM</td>
</tr>
<tr>
<td>0.5M EDTA</td>
<td>10 mL</td>
<td>f.10 mM</td>
</tr>
</tr>
</tbody>
</table>
<p>Procedure</p>
<p>1) Mix the reagents.</p>
<p>2) Anneal the mixture at XX °C for XX hours.</p>
<p>3) Analyze by 1 % agarose gel electrophoresis.</p>
<p>4) Stain the gel by EtBr for 30 minutes.</p>
<p>5) Take photo of the gel by LAS-4000.</p>
<br>
<a name="1-2"> </a> <br><br><br><br> <p class="small">1-2. Controlling polymerization</p>
<a name="1-3"> </a> <br><br><br><br> <p class="small">1-3. Putting the Monomer into a liposome</p> <br>
<a name="1-4"> </a><br><br> <a name="1-4-1"> </a><br><br>
<p class="small">1-4. Polymerization</p>
<p class="small"> 1-4-1. Polymerization in solution</p> <table class="sample_01"> <tbody> <tr> <th>Reagents</th> <th> </th> </tr> <tr> <td>Purified Monomer A</td> <td> µL</td> </tr> <tr> <td>Purified Monomer B</td> <td> µL</td> </tr> </tbody> </table> <p>Procedure</p> <p>1) Make monomers as shown in “Making DNA origami monomers”.</p> <p>2) Purify each monomer solution by spin column.</p> <p>3) Mix the reagents.</p> <p>4) Incubate at room temperature for XX hours.</p> <p>5) Analyze mixture by 1 % agarose gel electrophoresis (100V, 80 min).</p> <a name="1-4-2"> </a><br> <p>6) Stain the gel by EtBr for 30 minutes.</p> <p>7) Take photographs of the gel by LAS-4000.</p>
<p class="small"> 1-4-2. Polymerization in a liposome
<a name="2"> </a><br><br><br> <a name="2-1"> </a> <h1 class="title"><a name="contents"> 2. Receptor</a></h1>
<p class="small">2-1. Making the Receptor</p> <table class="sample_01"> <tbody> <tr> <th>Reagents</th> <th> </th> </tr> <tr> <td>M13mp18ss</td> <td> </td> </tr> <tr> <td>Staple mix</td> <td> </td> </tr> <tr> <td>10 × barrel buffer¹</td> <td> </td> </tr> <tr> <td>MQ</td> <td> </td> </tr> </body> </table> <table class="sample_03"> <tbody> <tr> <th>¹10 × barrel buffer (f:10ml)</th> <th> </th> </tr> <tr> <td>1M Tris Baric</td> <td>0.5 µL</td> </tr> <tr> <td>0.5M EDTA</td> <td>200 µL</td> </tr> <tr> <td>1M MgCl<sub>2</sub></td> <td>2 mL</td> </tr> <tr> <td>5M NaCl</td> <td>100 µL</td> </tr> <tr> <td>MQ</td> <td>7.2 mL</td> </tr> </tbody> </table> <p>Procedure</p> <p>1) Mix the solutions.</p> <p> 2) The mixture was annealed at 47.5oC for 4 hours.</p> <p><a name="2-2"> </a>3) Purified with spin column.</p> <p>4) Analyzed by 1% agarose gel electrophoresis (100V, 40 min).</p> <br>
<p class="small">2-2. Penetration into a liposome</p>
<a name="2-3"> </a> <br><br> <a name="2-3-1"> </a> <br><br>
<p class="small">2-3. Dimerization</p>
<p class="small"> 2-3-1. Dimerization in solution</p> <table class="sample_01"> <tbody> <tr> <th>Reagents</th> <th> </th> </tr> <tr> <td>Receptor E</td> <td> </td> </tr> <tr> <td>Receptor A</td> <td> </td> </tr> <tr> <td>Thrombin (1.09µM)</td> <td> </td> </tr> <tr> <td>Loading-dye</td> <td> </td> </tr> </tbody> </table> <p>Procedure</p> <p>1) Mix the solutions, and incubated 1 hour at room temperature.</p> <a name="2-3-2"> </a> <p>2) Analyzed by 1% agarose gel electrophoresis (100V, 80 min).</p>
<br> <p class="small"> 2-3-2. Dimerization on a liposome</p>
<a name="2-4"> </a> <br><br> <a name="2-4-1"> </a> <br><br> <p class="small">2-4. Emission of polymerization initiator by dimerization</p>
<p class="small"> 2-4-1. Emission by dimerization in solution</p>
<a name="2-4-2"> </a> <br><br><br><br> <p class="small"> 2-4-2. Emission by dimerization on a liposome</p>
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