Biomod/2014/Kashiwa/Protocol
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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="Century,Arial" font size="20pt">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> 1. Polymerization <ul style="list-style:none;"> <li> 1-1. Making the monomer </li> <li> 1-2. Polymerization in solution <ul style="list-style:none;"> <li> 1-2-1. <li> 1-2-2. </ul> </li> <li> 1-3. Putting monomer into a liposome </li> <li> 1-4. Polymerize in a liposome </li> <li> 1-5. Deformation of a liposome </li> </ul> </li> <li> 2. Receptor <ul style="list-style:none"> <li> 2-1. Making the receptor </li> <li> 2-2. Penetration into a liposome </li> <li> 2-3. Dimerization <ul style="list-style:none"> <li> 2-3-1. Dimerization in solution </li> <li> 2-3-2. Dimerization on a liposome </li> </ul> </li> <li> 2-4. Emission of polymerization initiator by dimerization <ul style="list-style:none"> <li> 2-4-1. Emission by dimerization in solution </li> <li> 2-4-2. Emmision by dimerization on a liposome </li> </ul> </li> </ul> </ul> </div>
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<br> <h1 class="title"><a name="contents"> 1. Polymerization</a></h1> <br> 1-1. Making the monomer <table class="sample_01"> <tbody> <tr> <th>Reagent</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>
<table class="sample_02"> <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>
Reagents (f. 60μL)
Staple mix 17.5 μL
M13mp18ss 18.3 μL
TE*1 18.2 μL
10 x tile buffer*2 6.0 μL
- 1 TE (f. 50 mL)
1M Tris-HCl (pH 8.0) 0.5 mL (f. 10 mM)
0.5M EDTA 0.1 mL (f. 1 mM)
- 2 10 x tile buffer (f. 500 mL)
Mg(OAc)2 10.73 g f. 100 mM
1.0 M Tris-HCl (pH 7.5) 100 mL f. 200 mM
0.5 M EDTA 10mL f. 10 mM
1) Mix the reagents.
2) Anneal the mixture at XX °C for XX hours.
3) Analyze by 1 % agarose gel electrophoresis.
4) Stain the gel by EtBr for 30 minutes.
5) Take photo of the gel by LAS-4000.
2-3-1.Receptor monomers are assembled and heterodimerized by Thrombin.
【1:Assembly of receptor】(Barrel ver.) <table class="sample_01"> <tbody> <tr> <th>Reagent</th> <th> </th> </tr> <tr> <td>M13mp18ss</td> <td> </td> </tr> <tr> <td>Staple mix</td> <td> </td> </tr> <tr> <td>10x barrel buffer</td> <td> </td> <tr> <td>MQ</td> <td> </td> </tr> </tr> </tbody> </table>
- 10x barrel buffer (f:10ml)
1M Tris Baric 0.5μl
0.5M EDTA 200μl
1M MgCl2 2ml
5M NaCl 100μl
MQ 7.2ml
[Procedure]
1. Mix the solutions.
2. The mixture was annealed at 47.5oC for 4 hours.
3. Purified with spin column.
4. Analyzed by 1% agarose gel electrophoresis (100V, 40 min).
【2:Dimerization of hetero receptor by Thrombin】
[Reagent]
Monomer A (purified)
Monomer B (purified)
Thrombin (1.09μM)
Loading-dye
[Procedure]
1. Mix the solutions, and incubated 1 hour at room temperature.
2. Analyzed by 1% agarose gel electrophoresis (100V, 80 min).
<h1 class="title"><a name="contents"> 2. Receptor</a></h1>
挫折。また明日。
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