Biomod/2014/Kashiwa/Protocols: Difference between revisions

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 <ul id="menu">
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   <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>
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   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Project" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">PROJECT</span></a>
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       <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>
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   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Trial" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">&nbsp;EARLY TRIAL&nbsp;</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>
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   <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>
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   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Highlights" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">&nbsp;EXPERIMENT&nbsp;&nbsp;</span></a>
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       <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>
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   <li><a href="http://openwetware.org/wiki/Biomod/2014/Kashiwa/Discussion" onMouseOver="On('img1')" onMouseOut="Off()"><span style="font-size:12pt;">&nbsp;&nbsp;DISCUSSION&nbsp;</span></a>
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       <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>
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   <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>
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<body> <font face="Futura,Arial,Frutiger" font size="24px">PROTOCOL</font>

  <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">&nbsp; Contents：</p>
  </div>
  <div class="CollapsibleBoxBody" id="CBoxBody1">
     <ul style="list-style:none;">
        <li> <a href=#1> 1. The Sensing System: The Receptor </a>
        <ul style="list-style:none;">
           <li> <a href="#1-1">1-1. Preparing the components </a></li>
           <ul style="list-style:none;">
              <li> <a href="#1-1-1">1-1(a). Folding the Wall </a></li>
              <li> <a href="#1-1-2">1-1(b). Producing MISTIC </a></li>
              <li> <a href="#1-1-3">1-1(c). Designing the Activator </a></li>
           </ul>
           </li>
           <li> <a href="#1-2">1-2. Embedding the Wall into the liposome </a></li>
           <li> <a href="#1-3">1-3. Linking the Activator to the liposome </a></li>
           <li> <a href="#1-4">1-4. Combining the Wall and the Activator </a></li>
           <li> <a href="#1-5">1-5. Separating the Wall from the Activator </a></li>
        </ul>
        </li>
        
        <li> <a href="#2">2. The Moving System: The Motor </a>
        <ul style="list-style:none">
           <li> <a href="#2-1">2-1. Producing the Motor-Monomer </a>
           <ul style="list-style:none">
             <li> <a href="#2-1-1"> 2-1(a). Folding the Motor-Monomer body </a></li>
             <li> <a href="#2-1-2"> 2-1(b). Synthesizing divalent SA </a></li>
             <li> <a href="#2-1-3"> 2-1(c). Equipping the Motor-Monomer with divalent SA </a></li>
           </ul>
           </li>
           <li> <a href="#2-2">2-2. Deactivating and reactivating the binding capacity of streptavidin </a></li>
           <li> <a href="#2-3">2-3. Putting the Motor-Monomers into the liposome </a></li>
        </ul>
        </li>
     </ul>
  </div>

</div>

<br> <a name="1-1"> </a> <br> <a name="1-1-1"> </a> <h1 class="title"><a href=""> 1. The Sensing System: The Receptor</a></h1> <h1 class="sub">1-1. Preparing the components</h1> <h1 class="big">1-1(a). Folding the Wall</h1>

<table class="sample_01"><caption>Reagents (f. 10µL)</caption> <tbody> <tr><th>Staple mix</th><td>5 µL</td></tr> <tr><th>M13mp18ss</th><td>4 µL</td></tr> <tr><th>10 x monomer buffer*1</th><td>1 µL</td></tr> </tbody></table> <p class="paragraph">*1 10 x monomer buffer (100 mL) <p class="small">1.0 M Tris-HCl (pH 8.0) 10mL</p> <p class="small">1.0 M MgCl<sub>2</sub> (f. 150 mM) 15mL</p> <p class="small">1.0 M NaCl (f. 25 mM) 2.5mL</p></p>

<p class="paragraph"> <p class="menu">1) Mix the reagents.</p> <p class="menu">2) Anneal as follows</p> <p class="menu">85°C, 5 min till 49.3°C, 2h till 4°C, forever</p> <p class="menu">Analyze by 1 % agarose gel electrophoresis.</p> <p class="menu">Stain the gel by EtBr for 30 minutes.</p> <p class="menu">Take photo of the gel by LAS-4000.</p></p>

<h1 class="big">1-1(b). Producing MISTIC</h1>

<h1 class="big">1-1(c). Designing the Activator</h1> <p class="paragraph"><b>(i) Evaluation of Lambda Exonuclease activity</b></p> <p class="paragraph">Procedure <p class="menu">1) Two ssDNA mixtures (a and a’ mixture ,and b and b’ mixture) were annealed at 65℃ for 5 minutes.</p> <p class="menu">2) The two dsDNA reagents were mixed.</p> <p class="menu">3) The dsDNA mixture and Lambda Exonuclease were mixed and incubated at 37°C.</p> <p class="menu">4) The mixture was analyzed by 20% Native-PAGE.</p> <p class="menu">5) The gel was stained by SYBR Gold for 15 minutes.</p> <p class="menu">6) The gel was taken a photo by LAS-4000. </p>

<table class="sample_01"> <caption>Reaction Reagent</caption> <tbody> <tr><th>dsDNA mixture</th> <td>4µL</td></tr> <tr><th>Lambda Exonuclease</th><td>5µL</td></tr> <tr><th>10×reaction buffer</th><td>1µL</td></tr> </tbody></table>

<p class="paragraph"> 10×reaction buffers in each experiment are as below. <p class="small">1) 670mM Glycine-KOH(pH7.5 8.0, 8.5, 9.0, 9.4), 2.5mM MgCl<sub>2</sub>, 50&mibro;g/ml BSA <p class="small">2) 670mM Glycine-KOH(pH8.0), 2.5, 5.0, 7.5, 10.0, 12.5mM MgCl<sub>2</sub>, 50µg/ml BSA </p></p>

<p class="paragraph"><b>(ii) Oligo-modification of Lambda-Exonuclease</b></p> <table class="sample_01"> <caption>Reagent</caption> <tbody> <tr><th>0.2M KPO<sub>4</sub>(pH7.8)</th><td>5µL</td></tr> <tr><th>200µM Cy3-modified oligo</th><td>10µL</td></tr> <tr><th>30mM BS(PEG)<sub>9</sub></th> <td>2µL</td></tr></tbody></table>

<p class="paragraph"> Procedure <p class="menu">1) The solution was mixed.</p> <p class="menu">2) The mixture was incubated at room temperature for 5 minutes.</p> <p class="menu">3) The solution was refines by NAP-5 column.</p> <p class="menu">4) Lambda Exonuclease and refined oligo that are equimolecular amount were mixed.</p> <p class="menu">5) The mixture was incubated on ice for 3 hours.</p> <p class="menu">6) The solution was analyzed by 20% Native-PAGE and 20% SDS-PAGE.</p> <p class="menu">7) The Native-PAGE gel was taken a photo by LAS-4000.</p> <p class="menu">8) The SDS-PAGE gel was stained by SYPRO Orange for 60 minutes and taken a photo by LAS-400. </p></p>

<p class="paragraph"><b>(iii)Oligo-modification of HindⅢ</b></p> <p class="paragraph">Exchange-buffer</p> <table class="sample_01"><caption>Reagents (f: 50ml)</caption> <tbody> <tr><th>0.2M HEPES (pH8.5)</th><td> 5ml</td></tr> <tr><th>5M NaCl </th><td>500ul</td></tr> <tr><th>1M DTT </th><td>50ul</td></tr> <tr><th>0.5M EDTA </th><td>10ul</td></tr> <tr><th>MQ up to 50ml</th></tbody></table>

<p class="paragraph"> Reagents for Oligo-BS(PEG)<sub>9</sub> <p class="small">0.2M KPO<sub>4</sub> 5ul</p> <p class="small">200uM cy3-Oligonucleotide 10ul</p> <p class="small">20mM BS(PEG)<sub>9</sub></p> </p>

<p class="paragraph"> Reagents for Oligo- BS(PEG)<sub>9</sub>-HindⅢ <p class="small">Fraction 20ul</p> <p class="small">HindⅢ 20ul</p></p>

<h1 class="sub">1-2. Embedding the Wall into the liposome</h1> <p class="paragraph"><b>(i) Hybridization of cholesterol oligonucleotide with Wall</b></p> <p class="paragraph"> In this experiment, we optimized the concentration of the cholesterol oligonucleotide and the number of handles, which is necessary for wall penetration into liposome. The result was assayed by 1% agarose gel electrophoresis.</p> <table class="sample_01"> <caption>Reagents</caption> <tbody> <tr><th>Purified Wall (0.03µM)</th><td>6µL</td></tr> <tr><th>Cholesterol oligomer </th><td>1.5µL</td></tr> </tbody> </table>

<p class="paragraph">Sample2 and 3 do not have handles for the cholesterol oligonucleotide to hybridize. Sample 5 and 6 have 4, sample 8 and 9 have 10, sample 11 and 12 have handles for the cholesterol oligonucleotide to hybridize.</p> <table class="sample_01"> <caption> Concentration of cholesterol oligonucleotide</caption> <tbody> <tr><th> Sample2_1, 3_1, 5_1, 6_1</th><td>0.24µM</td></tr> <tr><th>Sample2_2, 3_2, 5_2, 6_2</th><td>0.48µM</td></tr> <tr><th>Sample2_3, 3_3, 5_3, 6_3</th><td>2.4µM</td></tr> <tr><th>Sample2_4, 3_4, 5_4, 6_4</th><td>4.8µM</td></tr> <tr><th>Sample8_1, 9_1</th><td>0.60µM</td></tr> <tr><th>Sample8_2, 9_2</th><td>1.2µM</td></tr> <tr><th>Sample8_3, 9_3</th><td>6.0µM</td></tr> <tr><th>Sample8_4, 9_4</th><td>12µM</td></tr> <tr><th>Sample11_1, 12_1</th><td>1.14µM</td></tr> <tr><th>Sample11_2, 12_2</th><td>2.28µM</td></tr> <tr><th>Sample11_3, 12_3</th><td>11.4µM</td></tr> <tr><th>Sample11_4, 12_4</th><td>22.8µM</td></tr> </tbody> </table> <p class="paragraph">Procedures</p> <p class="paragraph">Reagents are mixed and incubated for 60 minutes at room temperature.</p>

<p class="paragraph"><b>(ii) Putting the Wall into the liposome</b></p> <p class="paragraph">Same as <a href="#2-3">2-3. Putting the Monomer into the liposome</a>.</p>

<h1 class="sub">1-3. Linking the Activator to the liposome</h1> <h1 class="sub">1-4. Combining the Wall and the Activator</h1> <h1 class="sub">1-5. Separating the Wall from the Activator</h1>

<h1 class="title"><a href=""> 2. The Moving System: The Motor</a></h1> <h1 class="sub">2-1. Producing the Motor-Monomer</h1> <h1 class="big">2-1(a). Folding the Motor-Monomer body</h1>

<table class="sample_01"><caption>Reagents (f. 10µL)</caption> <tbody> <tr><th>Staple mix</th><td>5 µL</td></tr> <tr><th>M13mp18ss</th><td>4 µL</td></tr> <tr><th>10 x monomer buffer*1</th><td>1 µL</td></tr> </tbody></table> <p class="paragraph">*1 10 x monomer buffer (100 mL) <p class="small">1.0 M Tris-HCl (pH 8.0) 10mL</p> <p class="small">1.0 M MgCl<sub>2</sub> (f. 150 mM) 15mL</p> <p class="small">1.0 M NaCl (f. 25 mM) 2.5mL</p> </p>

<p class="paragraph"> <p class="menu">1) Mix the reagents.</p> <p class="menu">2) Anneal as follows</p> <p class="menu">85°C, 5 min till 49.3°C, 2h till 4°C, forever</p> <p class="menu">Analyze by 1 % agarose gel electrophoresis.</p> <p class="menu">Stain the gel by EtBr for 30 minutes.</p> <p class="menu">Take photo of the gel by LAS-4000.</p></p>

<h1 class="big">2-1(b). Synthesizing divalent SA</h1>

<h1 class="big">2-1(c). Equipping the Motor-Monomer with divalent SA</h1> <p class="paragraph"><b>(i) Modifying divalent SA with NHS</b></p>

<p class="paragraph">Procedure</p> <p class="paragraph"> <p class="menu">1) Exchange the buffer of divalent SA to the Exchange-buffer by NAP-5.</p> <p class="menu">2) Mix divalent SA with Cy5-NHS in tetraborate and incubate for 1 hour at room temperature.</p> <p class="menu">3) Add biotin-oligonucleotide to the mixture and incubate 30 minutes at room temperature.</p> <p class="menu">4) Analyze by Native-PAGE (10% Resolving Gel)</p>

<table class="sample_01"> <caption>Exchange-buffer</caption> <tbody> <tr> <th>0.2M HEPES (pH8.5)</th> <td>5ml</td></tr> <tr> <th>5M NaCl</th> <td>500µl</td></tr> <tr> <th>1M DTT</th> <td>50µl</td> </tr> <tr> <th>0.5M EDTA</th> <td>10µl</td> </tr> <tr> <th>MQ up to 50ml</th> </tr>

<table class="sample_01"> <caption>Reagents for SA and Cy5-NHS</caption> <tbody> <tr> <th>0.45µM divalent SA</th> <td>10µL</td> </tr> <tr> <th>Cy5-NHS</th>　<td>10µL</td> </tr> <tr> <th>tetraborate (pH 8.1)</th><td> 5µL</td> </tr> </tbody> </table>

<p class="paragraph">Reagents for Cy5-SA-biotin-oligonucleotide: <p class="small">Fraction 5µl</p> <p class="small">the threefold amounts of biotin-oligonucleotide 5µl</p> </p>

<p class="paragraph"><b>(ii) Click reaction</b></p> <p class="paragraph">Procedure</p> <p class="paragraph"> <p class="menu">1) Mix click reagent and NH2-modified oligonucleotide in tetraborate (pH8.1) and incubate for 1h at room temperature.</p> <p class="menu">2) Refine with QIA quick Nucleotide Column.</p> <p class="menu">3) Add 450µL of PNI buffer to the mixture.</p> <p class="menu">4) Apply to column.</p> <p class="menu">5)Centrifuge at 6krpm for 1minutes.</p> <p class="menu">6) Add 750 µL of buffer PE.</p> <p class="menu">7) Centrifuge at 6krpm for 1minutes.</p> <p class="menu">8) Add 750 µL of buffer PE.</p> <p class="menu">9) Centrifuge at 6krpm for 1minutes.</p> <p class="menu">10) Centrifuge at 13krpm for 1minutes.</p> <p class="menu">11) Put the column on new tube and add 33µL of MQ and incubate for 3 minutes.</p> <p class="menu">12) Centrifuge at 13krpm for 1minutes.</p> <p class="menu">13) Dilute the mixture with MQ to the appropriate concentrate.</p> <p class="menu">14) Mix one of Azide group and that of Alkyne group and incubate at room temperature.</p> <p class="menu">15) Analyze by 15% Urea gel electrophoresis.</p> </p>

<table class="sample_01"> <caption>Reagent of click reagent and NH2-modified oligonucleotide</caption> <tbody> <tr> <th>10mM click reagent</th> <td>7µL</td> </tr> <tr> <th>tetraborate(pH8.1)</th> <td>3µL</td> </tr> <tr> <th>2µg/µL　NH<sub>2</sub>-modified oligonucleotide</th> <td>2µL</td> </tr> </tbody> </table>

<table class="sample_01"> <caption>Reagent of click reaction</caption> <tbody> <tr> <th>6µM Azi-oligo</th> <td>5µL</td></tr> <tr> <th>6µM Alk-oligo</th> <td>5µL</td> </tr> <h1 class="sub">2-2. Deactivating and reactivating the binding activity of streptavidin</h1> <p class="paragraph"><b>(i) Make Biotin-Beads</b></p> <table class="sample_01"> <caption>Reagents (f: 20uL) </caption> <tbody> <tr><th>HEPES-KOH (pH8.5, f.90 mM)</th><td> 9.0 µL</td></tr> <tr><th>Biotin and amino modified Oligonucleotide (f: 65 µM)</th><td> 1 µL</td></tr> <tr><th>NHS-Beads (f.10 mg/ml)</th><td> 10 µL</td></tr> </tbody> </table>

<p class="paragraph"> Procedure</p> <p class="paragraph">Mix NHS-Beads with the oligonucleotide which has Biotin and amino group Remove the solvent.<p>

<p class="paragraph"><b>(ii) Deactivate and reactivate SA</b></p>

<table class="sample_01"><caption> Reagents for SA Deactivation (f: 10µL)</caption> <tbody> <tr><th>Ds DNA of Leader and Blocker (f: 5µM)</th> <td>2.0µL</td></tr> <tr><th>Divalent SA (f: 30µM)</th><td> 8.0µL</td></tr> <tr><th>Biotin-Beads</th><td> 0.2mg</td> <tr><th><sub>1</sub>Ds DNA (f: 10µL)</th></tr> <tr><th>Biotin modified Oligonucleotide (Leader) (f: 25µM)</th><td> 5.0µL</td></tr> <tr><th>Desthiobiotin modified Cy3-Oligonucleotide (Blocker) (f: 25µM)</th><td> 5.0µL</td></tr></tbody></table>

<table class="sample_01"><caption>Reagents for cutting dsDNA on SA (f: 15µL)</caption> <tbody> <tr><th>Purified Deactivation SA-dsDNA complex</th><td> 4µL</td></tr> <tr><th>HindⅢ (f: 6700U/ml)</th><td> 1uL</td></tr> <tr><th>10×NEB Buffer 2.1 </th><td>1.5µL</td></tr> <tr><th>MQ 8.5µL</th></tr></tbody></table>

<table class="sample_01"><caption>Reagents for cutting ds DNA on SA (f: 10uL)</caption> <tbody> <tr><th>Purified Deactivation SA-dsDNA complex</th><td> 2 µL</td></tr> <tr><th>Biotin modified Cy5-Oligonucleotide (Chaser) (f: 5 µM)</th><td> 2.0 µL</td></tr> <tr><th>MQ 6 µL</th></tr> </tbody></table>

<table class="sample_01"><caption>Reagents for reactivation of SA (f: 10 µL)</caption> <tbody> <tr> <th>dsDNA cutted SA-dsDNA complex（solution）</th> <td>8µL</td> </tr><tr> <th> Biotin modified Cy5-Oligonucleotide (Chaser) (f: 5 µM)</th> <td>2.0µL</td> </tr> </tbody> </table>

<p class="paragraph"> Reagents for Native-PAGE </p> <p class="paragraph">15 % Resolving Gel <p class="small">MQ 4.8 ml</p> <p class="small">30% Acrylamide mix 10 ml</p> <p class="small">1.5M Tris-HCl (pH 8.8) 5.0 ml</p> <p class="small">10 % APS 0.2 ml</p> <p class="small">TEMED 8.0 ul</p> </p>

<p class="paragraph"> 5 % Stacking Gel <p class="small">MQ 2.79 ml</p> <p class="small">30% Acrylamide mix 0.67 ml</p> <p class="small">1.0 M Tris-HCl (pH 6.8) 0.5 ml</p> <p class="small">10 % APS 40 ul</p> <p class="small">TEMED 4.0 ul</p>

<p class="paragraph">Procedure</p> <p class="menu">(i) Make Biotin-Beads</p> <p class="menu">(ii) Deactivate and reactivate SA</p> <p class="menu">1) Anneal dsDNA of Leader and Blocker (95°C, 3min to 4&degC, forever)</p> <p class="menu">2) Mix the dsDNA with divalent SA at 4℃ for 30min</p> <p class="menu">3) Remove SA which is not bound to the dsDNA by Biotin-Beads </p> <p class="menu">4) Cut the dsDNA bound to SA by HindⅢ</p> <p class="menu">5) Add Chaser to the SA of which the dsDNA was cut</p> </p>

<p class="paragraph">Analyze <p class="menu">1) Analyze by Native PAGE (15% Resolving Gel) <p class="menu">2) Observe with LAS-4000 and take pictures</p></p>

<h1 class="sub">2-3. Putting the Motor-Monomers into the liposome</h1> <p class="paragraph"><b>(i) Binding Motor-Monomers and Qdots</b>/p> <p class="paragraph">Reagents <p class="small">Motor-Monomers (unpurified) 80µL</p> <p class="small">Qdot 655 streptavidin (1µM) 6.4µL</p></p>

<p class="paragraph">Procedure <p class="menu">1) 80µL of Motor-Monomers and 6.4µL of Q-dot were mixed and incubated at room temperature for 30 minutes.</p> <p class="menu">2) The mixture was purified.</p></p>

<p class="paragraph"><b>(ii) Preparation of GUVs</b></p> <p class="paragraph">Reagents <p class="paragraph">Lipid mix (3mM) <p class="small">POPC 12mg</p> <p class="small">Paraffin 5mL</p></p>

<p class="paragraph">Outer solution <p class="small">Glucose (400mM) 250µL</p> <p class="small">Tris (50mM, pH 7.5) 250µL</p> </p>

<p class="paragraph">Emulsion mix <p class="small">Lipid mix (0.5mM) 500µL</p> <p class="small">Inner solution(200mM) 10µL</p> </p>

<p class="paragraph">Inner solution <p class="paragraph">1. GUV including DNA constructs <p class="small">Mercaptoethanol (4mM) 5.0µL</p> <p class="small">DNA constructs with Qdot 2.5µL</p> <p class="small">Sucrose (1M) 2.0µL</p> <p class="small">Tris (500mM, pH 7.5) 0.5µL</p></p> <p class="paragraph">2. GUV not including Motor-Monomers or Walls <p class="small">Sucrose (400mM) 5µL</p> <p class="small">Tris (50mM, pH 7.5) 5µ</p></p>

<p class="paragraph">Procedure <p class="menu">1) Take Glucose (500µL) into a tube as outer solution.</p> <p class="menu">2) Add Lipid mix (0.5mM) on the glucose solution carefully.</p> <p class="menu">3) Add Emulsion mix on the top and rapidly centrifuge at 4°C, 1800rpm for 10 minutes.</o> <p class="menu">4) Centrifuge again at 4°C, 5000rpm for 10 minutes.</p> <p class="menu">5) The upper layer is removed and GUVs are collected using pipetman. <p class="menu">6) 5µL of GUV was mixed with 0.5µL of Nile Red (10µM) and observed by confocal microscope. </p>

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Biomod/2014/Kashiwa/Protocols: Difference between revisions

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@@ Line 238: / Line 238: @@
 </p></p>
-<p class="paragraph"><b>(ii)Oligo-modification of Lambda-Exonuclease</b></p>
+<p class="paragraph"><b>(ii) Oligo-modification of Lambda-Exonuclease</b></p>
 <table class="sample_01">
 <caption>Reagent</caption>
@@ Line 354: / Line 354: @@
 <h1 class="big">2-1(c). Equipping the Motor-Monomer with divalent SA</h1>
-<p class="paragraph"><b>(i)  modify divalent SA with NHS</b></p>
+<p class="paragraph"><b>(i) Modifying divalent SA with NHS</b></p>
 <p class="paragraph">Procedure</p>
@@ Line 460: / Line 460: @@
 Remove the solvent.<p>
-<p class="paragraph"><b>(ii) Inactivate and reactivate SA</b></p>
+<p class="paragraph"><b>(ii) Deactivate and reactivate SA</b></p>
-<table class="sample_01"><caption> Reagents for SA inactivation (f: 10&micro;L)</caption>
+<table class="sample_01"><caption> Reagents for SA Deactivation (f: 10&micro;L)</caption>
 <tbody>
 <tr><th>Ds DNA of Leader and Blocker (f: 5&micro;M)</th> <td>2.0&micro;L</td></tr>
@@ Line 473: / Line 473: @@
 <table class="sample_01"><caption>Reagents for cutting dsDNA on SA (f: 15&micro;L)</caption>
 <tbody>
-<tr><th>Purified inactivation SA-dsDNA complex</th><td> 4&micro;L</td></tr>
+<tr><th>Purified Deactivation SA-dsDNA complex</th><td> 4&micro;L</td></tr>
 <tr><th>Hind&#8546; (f: 6700U/ml)</th><td> 1uL</td></tr>
 <tr><th>10×NEB Buffer 2.1 </th><td>1.5&micro;L</td></tr>
@@ Line 480: / Line 480: @@
 <table class="sample_01"><caption>Reagents for cutting ds DNA on SA (f: 10uL)</caption>
 <tbody>
-<tr><th>Purified inactivation SA-dsDNA complex</th><td> 2 &micro;L</td></tr>
+<tr><th>Purified Deactivation SA-dsDNA complex</th><td> 2 &micro;L</td></tr>
 <tr><th>Biotin modified Cy5-Oligonucleotide (Chaser) (f: 5 &micro;M)</th><td> 2.0 &micro;L</td></tr>
 <tr><th>MQ 6 &micro;L</th></tr>
@@ Line 515: / Line 515: @@
 <p class="paragraph">Procedure</p>
 <p class="menu">(i) Make Biotin-Beads</p>
-<p class="menu">(ii) Inactivate and reactivate SA</p>
+<p class="menu">(ii) Deactivate and reactivate SA</p>
 <p class="menu">1) Anneal dsDNA of Leader and Blocker (95&deg;C, 3min to 4&degC, forever)</p>
 <p class="menu">2) Mix the dsDNA with divalent SA at 4℃ for 30min</p>
@@ Line 539: / Line 539: @@
 <p class="menu">2) The mixture was purified.</p></p>
-<p class="paragraph"><b>Preparation of GUVs</b></p>
+<p class="paragraph"><b>(ii) Preparation of GUVs</b></p>
 <p class="paragraph">Reagents
 <p class="paragraph">Lipid mix (3mM)