Team:ZJU-China/Notebook

From 2011.igem.org

(Difference between revisions)
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     <td><div class="pagetitle" id="stra1"><a> <h3>Biobrick</h3>
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     <td><div class="pagetitle" id="stra1"><a> <h3>Biobrick</h3>  
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           <p>brief introduction</p></a></div></td>
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           <p>brief introduction</p></a></div></td>  
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     <td><div class="pagetitle" id="stra2"><a href="https://2011.igem.org/Team:ZJU-China/Notebook/Biofilm"> <h3>Biofilm Formation</h3>
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     <td><div class="pagetitle" id="stra2"><a href="https://2011.igem.org/Team:ZJU-China/Notebook/Biofilm"> <h3>Biofilm Formation</h3>  
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           <p>brief introduction</p></a></div></td>
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           <p>brief introduction</p></a></div></td>  
      
      
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                     <li id="kwick3"><a>week3</a></li>  
                     <li id="kwick4"><a>week4</a></li>  
                     <li id="kwick4"><a>week4</a></li>  
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                     <li id="kwick5"><a>week5</a></li>
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                     <li id="kwick5"><a>week5</a></li>  
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                     <li id="kwick6"><a>week6</a></li>
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                     <li id="kwick6"><a>week6</a></li>  
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                     <li id="kwick7"><a>week7</a></li>
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                     <li id="kwick7"><a>week7</a></li>  
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                     <li id="kwick8"><a>week8</a></li>
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                     <li id="kwick8"><a>week8</a></li>  
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                     <li id="kwick9"><a>week9</a></li>
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                     <li id="kwick9"><a>week9</a></li>  
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                     <li id="kwick10"><a>week10</a></li>
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                     <li id="kwick10"><a>week10</a></li>  
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                     <li id="kwick11"><a>week11</a></li>
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                     <li id="kwick11"><a>week11</a></li>  
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                     <li id="kwick12"><a>week12</a></li>
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                     <li id="kwick12"><a>week12</a></li>  
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                     <li id="kwick13"><a style="font-size:18pt;margin:1px;padding:1px;">after week12</a></li>
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                     <li id="afkwick12"><a style="font-size:18pt;margin:1px;padding:1px;">after week12</a></li>  
                 </ul>  
                 </ul>  
             </div>  
             </div>  
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<div class="bigblock"><div class="inner" id="week1">
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<div class="bigblock"><div class="inner" id="week1">  
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<div class="block" id="nsheet">
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<div class="block" id="nsheet">  
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<h3>Week1</h3><hr/>
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<h3>Week1</h3><hr/>  
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<table id="notesheet" width="650" border="1" cellspacing="0" cellpadding="1">
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<table id="notesheet" width="650" border="1" cellspacing="0" cellpadding="1">  
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   <tr><td width="76">Day</td><td width="349">Note</td></tr>
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   <tr><td width="76">Day</td><td width="349">Note</td></tr>  
    
    
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   <tr>
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   <tr>  
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     <td id="sheetleft">Jul.4th Monday</td>
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     <td id="sheetleft">Jul.4th Monday</td>  
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     <td><table id="intable" width="328" border="0" cellspacing="0" cellpadding="1">
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     <td><table id="intable" width="328" border="0" cellspacing="0" cellpadding="1">  
    
    
-
     <td width="128">• Aerobic cultivation of DH5α</td>
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     <td width="128">• Aerobic cultivation of DH5α</td>  
    
    
    
    
-
     <td width="196">• Preparation of apparatus for the formation of biofilm</td>
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     <td width="196">• Preparation of apparatus for the formation of biofilm</td>  
    
    
-
</table></td>
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</table></td>  
-
   </tr>
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   </tr>  
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   <tr>
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   <tr>  
     <td id="sheetleft">Jul 5th Tuesday
     <td id="sheetleft">Jul 5th Tuesday
-
</td>
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</td>  
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     <td>&nbsp;</td>
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     <td>&nbsp;</td>  
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   </tr>
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   </tr>  
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   <tr>
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   <tr>  
     <td id="sheetleft">Jul.6th
     <td id="sheetleft">Jul.6th
Wednesday
Wednesday
-
</td>
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</td>  
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     <td><table width="217" border="0" cellspacing="0" cellpadding="1">
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     <td><table width="217" border="0" cellspacing="0" cellpadding="1">  
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   <tr>
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   <tr>  
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     <td width="215">•Receiving  primers ordered previously</td>
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     <td width="215">•Receiving  primers ordered previously</td>  
      
      
-
   </tr>
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   </tr>  
-
</table>
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</table>  
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</td>
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</td>  
-
   </tr>
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   </tr>  
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   <tr>
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   <tr>  
     <td id="sheetleft">Jul.7th
     <td id="sheetleft">Jul.7th
Thursday
Thursday
-
</td>
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</td>  
-
     <td><table width="238" border="0" cellspacing="0" cellpadding="1">
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     <td><table width="238" border="0" cellspacing="0" cellpadding="1">  
-
   <tr>
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   <tr>  
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     <td width="200">• Preparation of the aliquot of the primers</td>
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     <td width="200">• Preparation of the aliquot of the primers</td>  
-
     <td width="200">• Something wrong with a shaking incubator</td>
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     <td width="200">• Something wrong with a shaking incubator</td>  
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   </tr>
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   </tr>  
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</table>
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</td>
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</td>  
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   </tr>
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   </tr>  
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   <tr>
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   <tr>  
     <td id="sheetleft">Jul.8th
     <td id="sheetleft">Jul.8th
Friday
Friday
-
</td>
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</td>  
-
     <td><table width="222" border="0" cellspacing="0" cellpadding="1">
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     <td><table width="222" border="0" cellspacing="0" cellpadding="1">  
-
       <tr>
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       <tr>  
-
         <td width="155">• Preparation of culture plates for the transformations</td>
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         <td width="155">• Preparation of culture plates for the transformations</td>  
-
       </tr>
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       </tr>  
-
     </table></td>
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     </table></td>  
-
   </tr>
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   </tr>  
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   <tr>
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   <tr>  
     <td id="sheetleft">Jul.9th
     <td id="sheetleft">Jul.9th
Saturday
Saturday
-
</td>
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</td>  
-
     <td><table width="313" border="0" cellspacing="0" cellpadding="1">
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     <td><table width="313" border="0" cellspacing="0" cellpadding="1">  
-
   <tr>
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   <tr>  
-
     <td width="127">• Preparation of culture plates for the transformations</td>
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     <td width="127">• Preparation of culture plates for the transformations</td>  
-
     <td width="182">• protocols of transformation</td>
+
     <td width="182">• protocols of transformation</td>  
-
   </tr>
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   </tr>  
-
</table>
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</table>  
-
</td>
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</td>  
-
   </tr>
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   </tr>  
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   <tr>
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   <tr>  
     <td id="sheetleft">Jul.10th
     <td id="sheetleft">Jul.10th
Sunday
Sunday
-
</td>
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</td>  
-
     <td><table width="246" border="0" cellspacing="0" cellpadding="1">
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     <td><table width="246" border="0" cellspacing="0" cellpadding="1">  
-
       <tr>
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       <tr>  
         <td>• Several colonies were picked up and cultivated in  5mL LB medium.
         <td>• Several colonies were picked up and cultivated in  5mL LB medium.
•Cryosectioning of biofilm  
•Cryosectioning of biofilm  
-
</td>
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</td>  
-
       </tr>
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       </tr>  
-
     </table></td>
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     </table></td>  
-
   </tr>
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   </tr>  
-
 
+
-
</table>
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</table>  
-
<p>&nbsp;</p>
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<p>&nbsp;</p>  
-
</div>
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</div>  
-
 
+
-
<div class="block" id="summ">
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<div class="block" id="summ">  
-
<h3 style="color:white;">Data&protocol</h3>
+
<h3 style="color:white;">Data&amp;protocol</h3>  
-
<hr/>
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<hr/>  
-
<div class="block" id="monday">
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<div class="block" id="monday">  
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<h3>Monday</h3>
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<h3>Monday</h3>  
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<hr/>
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<hr/>  
-
<img src="https://static.igem.org/mediawiki/2011/6/61/IMG_8382.jpg" style="margin-left:100px;"  alt="blanknote" />
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<img src="https://static.igem.org/mediawiki/2011/6/61/IMG_8382.jpg" style="margin-left:100px;"  alt="blanknote" />  
-
<p>There is no data in first week</p>
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<p>There is no data in first week</p>  
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</div>
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</div>  
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<p>&nbsp;</p>
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<p>&nbsp;</p>  
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</div>
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</div>  
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</div>
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</div>  
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<!-- 11111111111111111111111111111111111111111111111111 -->
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<!-- 11111111111111111111111111111111111111111111111111 -->  
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<div class="inner" id="week2">
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<div class="inner" id="week2">  
-
<div class="block" id="nsheet">
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<div class="block" id="nsheet">  
-
<h3>Week2</h3><hr/>
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<h3>Week2</h3><hr/>  
-
<table id="notesheet" width="650" border="1" cellspacing="0" cellpadding="1">
+
<table id="notesheet" width="650" border="1" cellspacing="0" cellpadding="1">  
-
   <tr><td width="76">Day</td><td width="349">Note</td></tr>
+
   <tr><td width="76">Day</td><td width="349">Note</td></tr>  
    
    
-
   <tr>
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   <tr>  
-
     <td id="sheetleft">Jul.11th Monday</td>
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     <td id="sheetleft">Jul.11th Monday</td>  
-
     <td><table id="intable" width="541" border="0" cellspacing="0" cellpadding="1">
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     <td><table id="intable" width="541" border="0" cellspacing="0" cellpadding="1">  
    
    
-
     <td width="128">•  Set up new LB culture plates with ampicillin and kanamycin</p></td>
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     <td width="128">•  Set up new LB culture plates with ampicillin and kanamycin</p></td>  
    
    
    
    
     <td width="196">•  
     <td width="196">•  
-
       ·Sterilization of Glycerol and <br />
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       ·Sterilization of Glycerol and <br />  
-
         ·Preparation of 25mg/mL kanamycin</td>
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         ·Preparation of 25mg/mL kanamycin</td>  
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   <td>•Transformation of the parts mentioned on Jul.9th for the second time</td>
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   <td>•Transformation of the parts mentioned on Jul.9th for the second time</td>  
-
   <td>•Observation the sections</td>
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   <td>•Observation the sections</td>  
-
</table></td>
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</table></td>  
-
   </tr>
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   </tr>  
-
   <tr>
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   <tr>  
     <td id="sheetleft">Jul 12th Tuesday
     <td id="sheetleft">Jul 12th Tuesday
-
</td>
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</td>  
-
     <td><table id="intable" width="560" border="0" cellspacing="0" cellpadding="1">
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     <td><table id="intable" width="560" border="0" cellspacing="0" cellpadding="1">  
    
    
-
     <td width="128">• Pick two colonies of each parts and cultivate them in LB medium</td>
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     <td width="128">• Pick two colonies of each parts and cultivate them in LB medium</td>  
    
    
    
    
-
     <td width="203"> •Transformation of three parts(20J,20H.22B from Kit plates of 2011 Distribution ) which are related to the degradation of Cellulose </td>
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     <td width="203"> •Transformation of three parts(20J,20H.22B from Kit plates of 2011 Distribution ) which are related to the degradation of Cellulose </td>  
-
   <td width="91">•·Mini  prep to isolate 10I,12I and 22M</td>
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   <td width="91">•·Mini  prep to isolate 10I,12I and 22M</td>  
-
   <td width="130">•Conservation of 10I,12I,22M and 11P</td>
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   <td width="130">•Conservation of 10I,12I,22M and 11P</td>  
-
</table></td>
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</table></td>  
-
   </tr>
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   </tr>  
-
   <tr>
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   <tr>  
     <td id="sheetleft">Jul.13th
     <td id="sheetleft">Jul.13th
Wednesday
Wednesday
-
</td>
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</td>  
-
     <td><table width="640" border="0" cellspacing="0" cellpadding="1">
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     <td><table width="640" border="0" cellspacing="0" cellpadding="1">  
-
   <tr>
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   <tr>  
-
     <td width="120">• Place the culture plate of 20J,20H and 22B in the fridge.</td>
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     <td width="120">• Place the culture plate of 20J,20H and 22B in the fridge.</td>  
     <td width="102">•Min prep to isolate 13K  
     <td width="102">•Min prep to isolate 13K  
•Conservation of 13K
•Conservation of 13K
-
</td>
+
</td>  
-
<td width="156">•Restriction digest of the parts(12I,10I,22M,13K) with EcoRI and PstI</td>
+
<td width="156">•Restriction digest of the parts(12I,10I,22M,13K) with EcoRI and PstI</td>  
-
<td width="95">•Gel electrophoresis to analyse restriction fragments</td>
+
<td width="95">•Gel electrophoresis to analyse restriction fragments</td>  
-
<td width="157">•Test the Tm of Primers CP1&CS,NP&NS with 13K. The result of Gel electrophoresis shows that 60.2℃ is the Tm of NS and NP, and 57.4℃ is the Tm of CS and CP1.</td>
+
<td width="157">•Test the Tm of Primers CP1&amp;CS,NP&amp;NS with 13K. The result of Gel electrophoresis shows that 60.2℃ is the Tm of NS and NP, and 57.4℃ is the Tm of CS and CP1.</td>  
-
   </tr>
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   </tr>  
-
</table>
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</table>  
-
</td>
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</td>  
-
   </tr>
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   </tr>  
-
   <tr>
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   <tr>  
     <td id="sheetleft">Jul.14th
     <td id="sheetleft">Jul.14th
Thursday
Thursday
-
</td>
+
</td>  
-
     <td><table width="618" border="0" cellspacing="0" cellpadding="1">
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     <td><table width="618" border="0" cellspacing="0" cellpadding="1">  
-
   <tr>
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   <tr>  
     <td >• Test the Tm of the YFP,RFP,tetR,Vgb and nirB primers.
     <td >• Test the Tm of the YFP,RFP,tetR,Vgb and nirB primers.
The result of Gel electrophoresis shows that the Tm for the primers of Vgb is 54℃ and the Tm for the primers of nirB is 55℃.The RCR of YFP,RFP and tetR failed.
The result of Gel electrophoresis shows that the Tm for the primers of Vgb is 54℃ and the Tm for the primers of nirB is 55℃.The RCR of YFP,RFP and tetR failed.
-
</td>
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</td>  
    
    
-
   </tr>
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   </tr>  
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</table>
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</table>  
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</td>
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</td>  
-
   </tr>
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   </tr>  
-
   <tr>
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   <tr>  
     <td id="sheetleft">Jul.15th
     <td id="sheetleft">Jul.15th
Friday
Friday
-
</td>
+
</td>  
-
     <td><table width="600" border="0" cellspacing="0" cellpadding="1">
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     <td><table width="600" border="0" cellspacing="0" cellpadding="1">  
-
       <tr>
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       <tr>  
-
         <td width="155"> • PCR(Phusion)<br/>
+
         <td width="155"> • PCR(Phusion)<br/>  
• Digest 10I, 22M, 13K
• Digest 10I, 22M, 13K
• Used wrong cutter, digestion again.
• Used wrong cutter, digestion again.
-
</td>
+
</td>  
-
<td>• Run the results of PCR and the first digestion. The annealing temperature of YFP needed change. The digestion results confirmed</td>
+
<td>• Run the results of PCR and the first digestion. The annealing temperature of YFP needed change. The digestion results confirmed</td>  
-
<td>• Run the digestion results of second time. The bands are confirmed.</td>
+
<td>• Run the digestion results of second time. The bands are confirmed.</td>  
-
       </tr>
+
       </tr>  
-
     </table></td>
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     </table></td>  
-
   </tr>
+
   </tr>  
-
   <tr>
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   <tr>  
     <td id="sheetleft">Jul.16th
     <td id="sheetleft">Jul.16th
Saturday
Saturday
-
</td>
+
</td>  
-
     <td><table width="620" border="0" cellspacing="0" cellpadding="1">
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     <td><table width="620" border="0" cellspacing="0" cellpadding="1">  
-
   <tr>
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   <tr>  
-
     <td width="127">• Cut the linearized pSB1k3 with E+P</td>
+
     <td width="127">• Cut the linearized pSB1k3 with E+P</td>  
-
     <td width="123">• Purify the digestion results of 22M, 13K, 10I</td>
+
     <td width="123">• Purify the digestion results of 22M, 13K, 10I</td>  
-
     <td width="133">• Confirm digestion of pSB1k3 by electrophoresis, then purification</td>
+
     <td width="133">• Confirm digestion of pSB1k3 by electrophoresis, then purification</td>  
-
     <td width="113">• Test Tm of YFP<br/>
+
     <td width="113">• Test Tm of YFP<br/>  
• ligation: 22M+10I, 13K+10I
• ligation: 22M+10I, 13K+10I
-
</td>
+
</td>  
<td width="114">• Tm of YFP is 54 degree
<td width="114">• Tm of YFP is 54 degree
• transform the ligation results.
• transform the ligation results.
-
</td>
+
</td>  
-
   </tr>
+
   </tr>  
-
</table>
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</table>  
-
</td>
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</td>  
-
   </tr>
+
   </tr>  
-
   <tr>
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   <tr>  
     <td id="sheetleft">Jul.17th
     <td id="sheetleft">Jul.17th
Sunday
Sunday
-
</td>
+
</td>  
-
     <td><table width="620" border="0" cellspacing="0" cellpadding="1">
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     <td><table width="620" border="0" cellspacing="0" cellpadding="1">  
-
       <tr>
+
       <tr>  
-
         <td>• PCR 22M<br/>
+
         <td>• PCR 22M<br/>  
• purify Vgb, RFP, RFP-tetR, YFP-tetR. Gibson PCR.
• purify Vgb, RFP, RFP-tetR, YFP-tetR. Gibson PCR.
-
</td>
+
</td>  
-
<td>• ligation the purified the fragments in yesterday.</td>
+
<td>• ligation the purified the fragments in yesterday.</td>  
-
<td>• 22M PCR<br/>
+
<td>• 22M PCR<br/>  
• Transform the ligation results.
• Transform the ligation results.
-
</td>
+
</td>  
-
       </tr>
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       </tr>  
-
     </table></td>
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     </table></td>  
-
   </tr>
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   </tr>  
-
 
+
-
</table>
+
</table>  
-
<p>&nbsp;</p>
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<p>&nbsp;</p>  
-
 
+
-
 
+
-
</div>
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</div>  
-
<div class="block" id="summ">
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<div class="block" id="summ">  
-
<h3 style="color:white;">Data&protocol</h3>
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<h3 style="color:white;">Data&amp;protocol</h3>  
-
<hr/>
+
<hr/>  
-
<div class="block" id="monday">
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<div class="block" id="monday">  
-
<h3>Jul.13th Wednesday</h3>
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<h3>Jul.13th Wednesday</h3>  
-
<hr/>
+
<hr/>  
-
<p><strong>Systems of restriction digestion with EcoRI and PstI</strong></p>
+
<p><strong>Systems of restriction digestion with EcoRI and PstI</strong></p>  
-
 
+
-
<table style="margin:15px;" width="110" border="1" cellspacing="0" cellpadding="1">
+
<table style="margin:15px;" width="110" border="1" cellspacing="0" cellpadding="1">  
-
   <tr>
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   <tr>  
-
     <td>Plasmid</td>
+
     <td>Plasmid</td>  
-
     <td>1μL (>100ng)</td>
+
     <td>1μL (>100ng)</td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
-
     <td>EcoRI</td>
+
     <td>EcoRI</td>  
-
     <td>1μL</td>
+
     <td>1μL</td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
-
     <td>PstI</td>
+
     <td>PstI</td>  
-
     <td>1μL</td>
+
     <td>1μL</td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
-
     <td>10×Buffer Tango</td>
+
     <td>10×Buffer Tango</td>  
-
     <td>2μL</td>
+
     <td>2μL</td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
-
   <td>ddH2O</td>
+
   <td>ddH2O</td>  
-
   <td>15μL</td>
+
   <td>15μL</td>  
-
   </tr>
+
   </tr>  
-
</table>
+
</table>  
-
<img  src="https://static.igem.org/mediawiki/2011/c/c3/0713program.png"  />
+
<img  src="https://static.igem.org/mediawiki/2011/c/c3/0713program.png"  />  
-
 
+
-
<p><strong>Temperature grad</strong><br/>56℃,57.4℃,60.2℃,62.9℃,64.3℃,65.8℃</p>
+
<p><strong>Temperature grad</strong><br/>56℃,57.4℃,60.2℃,62.9℃,64.3℃,65.8℃</p>  
-
 
+
-
<p>&nbsp;</p>
+
<p>&nbsp;</p>  
-
 
+
-
<p><strong>PCR system (test the Tm of the primers CP1&CS, NP&NS)</strong></p>
+
<p><strong>PCR system (test the Tm of the primers CP1&amp;CS, NP&amp;NS)</strong></p>  
-
 
+
-
<table style="margin:15px;" width="110" border="1" cellspacing="0" cellpadding="1">
+
<table style="margin:15px;" width="110" border="1" cellspacing="0" cellpadding="1">  
-
   <tr>
+
   <tr>  
-
     <td>10×Buffer</td>
+
     <td>10×Buffer</td>  
-
     <td> 2μL</td>
+
     <td> 2μL</td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
-
     <td>dNTPs</td>
+
     <td>dNTPs</td>  
-
     <td>0.5μL</td>
+
     <td>0.5μL</td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
-
     <td>primers  CP1  (NP)</td>
+
     <td>primers  CP1  (NP)</td>  
-
     <td>1μL</td>
+
     <td>1μL</td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
-
     <td>primers  CS    (NS)</td>
+
     <td>primers  CS    (NS)</td>  
-
     <td>1μL</td>
+
     <td>1μL</td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
-
     <td>Template </td>
+
     <td>Template </td>  
-
     <td>1μL</td>
+
     <td>1μL</td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
-
     <td>rTaq </td>
+
     <td>rTaq </td>  
-
     <td>0.2μL</td>
+
     <td>0.2μL</td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
-
     <td>H2O </td>
+
     <td>H2O </td>  
-
     <td>14.5μL</td>
+
     <td>14.5μL</td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
-
     <td>Total</td>
+
     <td>Total</td>  
-
     <td> 20μL</td>
+
     <td> 20μL</td>  
-
   </tr>
+
   </tr>  
-
</table>
+
</table>  
-
 
+
-
</div>
+
</div>  
-
<div class="block" id="tuesday">
+
<div class="block" id="tuesday">  
-
<h3>Jul.14th Thursday</h3>
+
<h3>Jul.14th Thursday</h3>  
-
<hr/>
+
<hr/>  
-
<p>PCR systems(test the Tm of the primers for YFP,RFP,tetR,Vgb and nirB )</p>
+
<p>PCR systems(test the Tm of the primers for YFP,RFP,tetR,Vgb and nirB )</p>  
-
<table width="110" border="1" cellspacing="0" cellpadding="1">
+
<table width="110" border="1" cellspacing="0" cellpadding="1">  
-
   <tr>
+
   <tr>  
-
     <td>10×Buffer</td>
+
     <td>10×Buffer</td>  
-
     <td> 2μL</td>
+
     <td> 2μL</td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
-
     <td>dNTPs</td>
+
     <td>dNTPs</td>  
-
     <td>0.5μL</td>
+
     <td>0.5μL</td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
-
     <td>primers F  </td>
+
     <td>primers F  </td>  
-
     <td>0.5μL</td>
+
     <td>0.5μL</td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
-
     <td>primers R </td>
+
     <td>primers R </td>  
-
     <td>0.5μL</td>
+
     <td>0.5μL</td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
-
     <td>Template </td>
+
     <td>Template </td>  
-
     <td>1μL</td>
+
     <td>1μL</td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
-
     <td>Taq </td>
+
     <td>Taq </td>  
-
     <td>0.2μL</td>
+
     <td>0.2μL</td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
-
     <td>H2O </td>
+
     <td>H2O </td>  
-
     <td>15.5μL</td>
+
     <td>15.5μL</td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
-
     <td>Total</td>
+
     <td>Total</td>  
-
     <td> 20μL</td>
+
     <td> 20μL</td>  
-
   </tr>
+
   </tr>  
-
</table>
+
</table>  
-
<p>&nbsp;</p>
+
<p>&nbsp;</p>  
-
<img  src="http://ung.igem.org/wiki/images/2/20/0714program.png"  />
+
<img  src="http://ung.igem.org/wiki/images/2/20/0714program.png"  />  
-
<p>Temperature grad and the results:</p>
+
<p>Temperature grad and the results:</p>  
-
<img  src="https://static.igem.org/mediawiki/2011/6/6b/0714tempera.png"  />
+
<img  src="https://static.igem.org/mediawiki/2011/6/6b/0714tempera.png"  />  
-
<p>vgb&nirB pcr test:</p>
+
<p>vgb&amp;nirB pcr test:</p>  
-
<img  src="https://static.igem.org/mediawiki/2011/7/74/20110714_vgb%26nirB_pcr_test.jpg"  />
+
<img  src="https://static.igem.org/mediawiki/2011/7/74/20110714_vgb%26nirB_pcr_test.jpg"  />  
-
<p>&nbsp;</p>
+
<p>&nbsp;</p>  
-
</div>
+
</div>  
-
<p>&nbsp;</p>
+
<p>&nbsp;</p>  
-
</div>
+
</div>  
-
</div>
+
</div>  
-
<!-- 2222222222222222222222222222222222222222222 -->
+
<!-- 2222222222222222222222222222222222222222222 -->  
-
<div class="inner" id="week3">
+
<div class="inner" id="week3">  
-
<div class="block" id="nsheet">
+
<div class="block" id="nsheet">  
-
<h3>Week3</h3><hr/>
+
<h3>Week3</h3><hr/>  
-
 
+
-
<table id="notesheet" width="650" border="1" cellspacing="0" cellpadding="1">
+
<table id="notesheet" width="650" border="1" cellspacing="0" cellpadding="1">  
-
   <tr><td width="76">Day</td><td width="349">Note</td></tr>
+
   <tr><td width="76">Day</td><td width="349">Note</td></tr>  
-
   <tr>
+
   <tr>  
-
     <td id="sheetleft">Jul.18th Monday</td>
+
     <td id="sheetleft">Jul.18th Monday</td>  
-
     <td><table id="intable" width="541" border="0" cellspacing="0" cellpadding="1">
+
     <td><table id="intable" width="541" border="0" cellspacing="0" cellpadding="1">  
    
    
-
     <td width="128">• Genome extraction  of E.coli </td>
+
     <td width="128">• Genome extraction  of E.coli </td>  
    
    
    
    
-
     <td width="196">•  PCR of NirB<br />
+
     <td width="196">•  PCR of NirB<br />  
-
         •  cut 10I with X+P, cut 22M, 13K with E+S, cut pSB1k3 with E+P</td>
+
         •  cut 10I with X+P, cut 22M, 13K with E+S, cut pSB1k3 with E+P</td>  
-
   <td>• Ligate: 10I+13K, 10I+22M <br/>
+
   <td>• Ligate: 10I+13K, 10I+22M <br/>  
• Repeat NirB PCR
• Repeat NirB PCR
-
</td>
+
</td>  
-
   <td><p align="left">• Culture 11P<br />
+
   <td><p align="left">• Culture 11P<br />  
-
     • Miniprep 10I, 22M, 13K</p></td>
+
     • Miniprep 10I, 22M, 13K</p></td>  
-
</table></td>
+
</table></td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
     <td id="sheetleft">Jul 19th Tuesday
     <td id="sheetleft">Jul 19th Tuesday
-
</td>
+
</td>  
-
     <td><table id="intable" width="615" border="0" cellspacing="0" cellpadding="1">
+
     <td><table id="intable" width="615" border="0" cellspacing="0" cellpadding="1">  
    
    
-
       <td width="140">• mini-prep: 10I+13K,  10I+22M</td>
+
       <td width="140">• mini-prep: 10I+13K,  10I+22M</td>  
    
    
    
    
-
     <td width="213"> •insert 10I+13K, 10I+22M into pSB1k3 </td>
+
     <td width="213"> •insert 10I+13K, 10I+22M into pSB1k3 </td>  
-
   <td width="110">•transform: 5E, 3C, 7C, 1K, 1I from the distribution plate</td>
+
   <td width="110">•transform: 5E, 3C, 7C, 1K, 1I from the distribution plate</td>  
-
   <td width="144">•transform 10I+22M, 10I+13K</td>
+
   <td width="144">•transform 10I+22M, 10I+13K</td>  
-
</table></td>
+
</table></td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
     <td id="sheetleft">Jul.20th
     <td id="sheetleft">Jul.20th
Wednesday
Wednesday
-
</td>
+
</td>  
-
     <td><table width="640" border="0" cellspacing="0" cellpadding="1">
+
     <td><table width="640" border="0" cellspacing="0" cellpadding="1">  
-
   <tr>
+
   <tr>  
-
     <td width="170">•Colonies of 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I confirmed</td>
+
     <td width="170">•Colonies of 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I confirmed</td>  
     <td width="102">• Gibson PCR
     <td width="102">• Gibson PCR
-
</td>
+
</td>  
-
<td >•Colony PCR 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I</td>
+
<td >•Colony PCR 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I</td>  
-
<td width="95">•PCR NirB</td>
+
<td width="95">•PCR NirB</td>  
-
   </tr>
+
   </tr>  
-
</table>
+
</table>  
-
</td>
+
</td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
     <td id="sheetleft">Jul.21th
     <td id="sheetleft">Jul.21th
Thursday
Thursday
-
</td>
+
</td>  
-
     <td><table width="618" border="0" cellspacing="0" cellpadding="1">
+
     <td><table width="618" border="0" cellspacing="0" cellpadding="1">  
-
   <tr>
+
   <tr>  
     <td >•PCR verification of 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I
     <td >•PCR verification of 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I
-
</td>
+
</td>  
<td>• Gibson PCR
<td>• Gibson PCR
• Run the results of PCR verification. Bands confirmed.
• Run the results of PCR verification. Bands confirmed.
-
</td>
+
</td>  
   <td>• Culture the 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I
   <td>• Culture the 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I
• Purification of Gibson PCR results
• Purification of Gibson PCR results
-
</td>
+
</td>  
-
   </tr>
+
   </tr>  
-
</table>
+
</table>  
-
</td>
+
</td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
     <td id="sheetleft">Jul.22th
     <td id="sheetleft">Jul.22th
Friday
Friday
-
</td>
+
</td>  
-
     <td><table width="627" border="0" cellspacing="0" cellpadding="1">
+
     <td><table width="627" border="0" cellspacing="0" cellpadding="1">  
-
       <tr>
+
       <tr>  
         <td width="117"> • PCR amplification of Gibson assembly results
         <td width="117"> • PCR amplification of Gibson assembly results
-
</td>
+
</td>  
-
<td width="144">• Mini prep: 22M+10I, 22.Presever in -20<br/>
+
<td width="144">• Mini prep: 22M+10I, 22.Presever in -20<br/>  
-
• Medi prep: 1K,1I,3C,5E,7C<br/>
+
• Medi prep: 1K,1I,3C,5E,7C<br/>  
• Gibson Assembly fail.
• Gibson Assembly fail.
-
</td>
+
</td>  
-
<td width="164">• PCR NirB by Phusion<br/>
+
<td width="164">• PCR NirB by Phusion<br/>  
-
•Repeat PCR by changing Pnibr to Gnirbr<br/>
+
•Repeat PCR by changing Pnibr to Gnirbr<br/>  
•Cut the mini and medi prep results with E
•Cut the mini and medi prep results with E
-
</td>
+
</td>  
-
<td width="194">•Run the results of PCR and digestion. Fail in PCR of NirB, succeed in 10I+3K and 10I+22M ligation.</td>
+
<td width="194">•Run the results of PCR and digestion. Fail in PCR of NirB, succeed in 10I+3K and 10I+22M ligation.</td>  
-
       </tr>
+
       </tr>  
-
     </table></td>
+
     </table></td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
     <td id="sheetleft">Jul.23th
     <td id="sheetleft">Jul.23th
Saturday
Saturday
-
</td>
+
</td>  
-
     <td><table width="620" border="0" cellspacing="0" cellpadding="1">
+
     <td><table width="620" border="0" cellspacing="0" cellpadding="1">  
-
   <tr>
+
   <tr>  
     <td >•Double digestion of 13K+10I, 22M+10I, pSB1c3
     <td >•Double digestion of 13K+10I, 22M+10I, pSB1c3
-
 
+
-
</td>
+
</td>  
-
     <td >• Fail in purification of Medi prep</td>
+
     <td >• Fail in purification of Medi prep</td>  
-
     <td >• PCR NirB<br/>
+
     <td >• PCR NirB<br/>  
• Ligate NirB+13K+10I, Vgb+22M+10I
• Ligate NirB+13K+10I, Vgb+22M+10I
-
</td>
+
</td>  
-
 
+
-
   </tr>
+
   </tr>  
-
</table>
+
</table>  
-
</td>
+
</td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
     <td id="sheetleft">Jul.24th
     <td id="sheetleft">Jul.24th
Sunday
Sunday
-
</td>
+
</td>  
-
     <td><table width="620" border="0" cellspacing="0" cellpadding="1">
+
     <td><table width="620" border="0" cellspacing="0" cellpadding="1">  
-
       <tr>
+
       <tr>  
-
         <td width="244">• Gibson assembly: NirB+RFP+tetR<br/>
+
         <td width="244">• Gibson assembly: NirB+RFP+tetR<br/>  
• Cut results of Gibson assembly and pSB1c3.
• Cut results of Gibson assembly and pSB1c3.
-
 
+
-
</td>
+
</td>  
-
<td width="164">• Purify the ligation results in yesterday</td>
+
<td width="164">• Purify the ligation results in yesterday</td>  
<td width="206">• ligate with backbone
<td width="206">• ligate with backbone
• Culture 1I, 1K, 3C, 5E, 7C, 11P
• Culture 1I, 1K, 3C, 5E, 7C, 11P
-
 
+
-
</td>
+
</td>  
-
       </tr>
+
       </tr>  
-
     </table></td>
+
     </table></td>  
-
   </tr>
+
   </tr>  
-
 
+
-
</table>
+
</table>  
-
<p>&nbsp;</p>
+
<p>&nbsp;</p>  
-
</div>
+
</div>  
-
<div class="block" id="summ">
+
<div class="block" id="summ">  
-
<h3 style="color:white;">Data&protocol</h3>
+
<h3 style="color:white;">Data&amp;protocol</h3>  
-
<hr/>
+
<hr/>  
-
<div class="block" id="monday">
+
<div class="block" id="monday">  
-
<h3>Monday</h3>
+
<h3>Monday</h3>  
-
<hr/>
+
<hr/>  
-
<p>WEEK3</p>
+
<p>WEEK3</p>  
-
</div>
+
</div>  
-
<div class="block" id="tuesday">
+
<div class="block" id="tuesday">  
-
<h3>Tuesday</h3>
+
<h3>Tuesday</h3>  
-
<hr/>
+
<hr/>  
-
<p>bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb</p></div>
+
<p>bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb</p></div>  
-
<p>&nbsp;</p>
+
<p>&nbsp;</p>  
-
</div>
+
</div>  
-
</div>
+
</div>  
-
<!-- 3333333333333333333333333333333333333 -->
+
<!-- 3333333333333333333333333333333333333 -->  
-
<div class="inner" id="week4">
+
<div class="inner" id="week4">  
-
<div class="block" id="nsheet">
+
<div class="block" id="nsheet">  
-
<h3>Week4</h3><hr/>
+
<h3>Week4</h3><hr/>  
-
 
+
-
<table id="notesheet" width="650" border="1" cellspacing="0" cellpadding="1">
+
<table id="notesheet" width="650" border="1" cellspacing="0" cellpadding="1">  
-
   <tr><td width="76">Day</td><td width="349">Note</td></tr>
+
   <tr><td width="76">Day</td><td width="349">Note</td></tr>  
    
    
-
   <tr>
+
   <tr>  
-
     <td id="sheetleft">Jul.25th Monday</td>
+
     <td id="sheetleft">Jul.25th Monday</td>  
-
     <td><table id="intable" width="541" border="0" cellspacing="0" cellpadding="1">
+
     <td><table id="intable" width="541" border="0" cellspacing="0" cellpadding="1">  
    
    
-
     <td width="128">• Transform Pnirb+13k+10I+pSBK3-2</td>
+
     <td width="128">• Transform Pnirb+13k+10I+pSBK3-2</td>  
    
    
    
    
-
     <td >• Place the culture in 4 degree</td>
+
     <td >• Place the culture in 4 degree</td>  
   <td>• Medi-and mini-prep of five cultre
   <td>• Medi-and mini-prep of five cultre
-
</td>
+
</td>  
-
   <td>•Gibson Assembly confirmation by gel</td>
+
   <td>•Gibson Assembly confirmation by gel</td>  
-
</table></td>
+
</table></td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
     <td id="sheetleft">Jul. 26th Tuesday
     <td id="sheetleft">Jul. 26th Tuesday
-
</td>
+
</td>  
-
     <td><table id="intable" width="615" border="0" cellspacing="0" cellpadding="1">
+
     <td><table id="intable" width="615" border="0" cellspacing="0" cellpadding="1">  
    
    
-
       <td width="140">• Cut Pnirb, Pvgb,(E+S)<br/>
+
       <td width="140">• Cut Pnirb, Pvgb,(E+S)<br/>  
• Cut 10I+13K, 10I+22M.(X+P)
• Cut 10I+13K, 10I+22M.(X+P)
-
</td>
+
</td>  
    
    
    
    
-
     <td width="181" > • Ligation: vgb+10I+22M, nirB+10I+13K,</td>
+
     <td width="181" > • Ligation: vgb+10I+22M, nirB+10I+13K,</td>  
-
   <td width="142">• Ligation: pSBK13+vgb+10I+22M, pSBK13+nirB+10I+13K</td>
+
   <td width="142">• Ligation: pSBK13+vgb+10I+22M, pSBK13+nirB+10I+13K</td>  
-
   <td width="144">• Transform the ligation results.</td>
+
   <td width="144">• Transform the ligation results.</td>  
-
</table></td>
+
</table></td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
     <td id="sheetleft">Jul.27th
     <td id="sheetleft">Jul.27th
Wednesday
Wednesday
-
</td>
+
</td>  
-
     <td><table width="640" border="0" cellspacing="0" cellpadding="1">
+
     <td><table width="640" border="0" cellspacing="0" cellpadding="1">  
-
   <tr>
+
   <tr>  
-
     <td width="170">• No colony on the plate<br/>
+
     <td width="170">• No colony on the plate<br/>  
• Gibson assembly
• Gibson assembly
-
</td>
+
</td>  
     <td width="184">• Run the results of Gibson assembly
     <td width="184">• Run the results of Gibson assembly
-
</td>
+
</td>  
-
<td width="280" >• Excise the bands, the purify the DNA<br/>
+
<td width="280" >• Excise the bands, the purify the DNA<br/>  
• Gibson Assenmly
• Gibson Assenmly
-
</td>
+
</td>  
-
 
+
-
   </tr>
+
   </tr>  
-
</table>
+
</table>  
-
</td>
+
</td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
     <td id="sheetleft">Jul.28th
     <td id="sheetleft">Jul.28th
Thursday
Thursday
-
</td>
+
</td>  
-
     <td><table width="618" border="0" cellspacing="0" cellpadding="1">
+
     <td><table width="618" border="0" cellspacing="0" cellpadding="1">  
-
   <tr>
+
   <tr>  
     <td >•2011 China Meet-up    @ Hefei
     <td >•2011 China Meet-up    @ Hefei
-
</td>
+
</td>  
-
 
+
-
   </tr>
+
   </tr>  
-
</table>
+
</table>  
-
</td>
+
</td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
     <td id="sheetleft">Jul.29th
     <td id="sheetleft">Jul.29th
Friday
Friday
-
</td>
+
</td>  
-
     <td><table width="627" border="0" cellspacing="0" cellpadding="1">
+
     <td><table width="627" border="0" cellspacing="0" cellpadding="1">  
-
       <tr>
+
       <tr>  
-
       <td >•2011 China Meet-up    @ Hefei</td>
+
       <td >•2011 China Meet-up    @ Hefei</td>  
-
       </tr>
+
       </tr>  
-
     </table></td>
+
     </table></td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
     <td id="sheetleft">Jul.30th
     <td id="sheetleft">Jul.30th
Saturday
Saturday
-
</td>
+
</td>  
-
     <td><table width="620" border="0" cellspacing="0" cellpadding="1">
+
     <td><table width="620" border="0" cellspacing="0" cellpadding="1">  
-
   <tr>
+
   <tr>  
      
      
-
 
+
-
   </tr>
+
   </tr>  
-
</table>
+
</table>  
-
</td>
+
</td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
     <td id="sheetleft">Jul.31th
     <td id="sheetleft">Jul.31th
Sunday
Sunday
-
</td>
+
</td>  
-
     <td><table width="620" border="0" cellspacing="0" cellpadding="1">
+
     <td><table width="620" border="0" cellspacing="0" cellpadding="1">  
-
       <tr>
+
       <tr>  
         <td width="244">• Culture 22M+10I, 13K+10I
         <td width="244">• Culture 22M+10I, 13K+10I
-
 
+
-
</td>
+
</td>  
-
<td width="164">• Cut a0H, 20J, 22B</td>
+
<td width="164">• Cut a0H, 20J, 22B</td>  
-
 
+
-
       </tr>
+
       </tr>  
-
     </table></td>
+
     </table></td>  
-
   </tr>
+
   </tr>  
-
 
+
-
</table>
+
</table>  
-
<p>&nbsp;</p>
+
<p>&nbsp;</p>  
-
</div>
+
</div>  
-
 
+
-
<div class="block" id="summ">
+
<div class="block" id="summ">  
-
 
+
-
<h3 style="color:white;">Data&protocol</h3>
+
<h3 style="color:white;">Data&amp;protocol</h3>  
-
<hr/>
+
<hr/>  
-
<div class="block" id="monday">
+
<div class="block" id="monday">  
-
<h3>Monday</h3>
+
<h3>Monday</h3>  
-
<hr/>
+
<hr/>  
-
<p>WEEK3</p>
+
<p>WEEK3</p>  
-
</div>
+
</div>  
-
<div class="block" id="tuesday">
+
<div class="block" id="tuesday">  
-
<h3>Tuesday</h3>
+
<h3>Tuesday</h3>  
-
<hr/>
+
<hr/>  
-
<p>bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb</p></div>
+
<p>bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb</p></div>  
-
<p>&nbsp;</p>
+
<p>&nbsp;</p>  
-
</div>
+
</div>  
-
</div>
+
</div>  
-
<!-- 444444444444444444444444444444444 -->
+
<!-- 444444444444444444444444444444444 -->  
-
<div class="inner" id="week5">
+
<div class="inner" id="week5">  
-
 
+
-
<div class="block" id="nsheet">
+
<div class="block" id="nsheet">  
-
<h3>Week5</h3><hr/>
+
<h3>Week5</h3><hr/>  
-
 
+
-
<table id="notesheet" width="650" border="1" cellspacing="0" cellpadding="1">
+
<table id="notesheet" width="650" border="1" cellspacing="0" cellpadding="1">  
-
   <tr><td width="76">Day</td><td width="349">Note</td></tr>
+
   <tr><td width="76">Day</td><td width="349">Note</td></tr>  
    
    
-
   <tr>
+
   <tr>  
     <td id="sheetleft">Aug.1st
     <td id="sheetleft">Aug.1st
Monday
Monday
-
</td>
+
</td>  
-
     <td><table id="intable" width="541" border="0" cellspacing="0" cellpadding="1">
+
     <td><table id="intable" width="541" border="0" cellspacing="0" cellpadding="1">  
    
    
     <td width="286">•Fusion PCR
     <td width="286">•Fusion PCR
Vgb+YFP+tetR, NirB+RFP+tetR,
Vgb+YFP+tetR, NirB+RFP+tetR,
-
</td>
+
</td>  
    
    
    
    
-
     <td width="251" >•PCR: NirB, Vgb</td>
+
     <td width="251" >•PCR: NirB, Vgb</td>  
    
    
-
</table></td>
+
</table></td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
     <td id="sheetleft">Aug.2nd
     <td id="sheetleft">Aug.2nd
Tuesday
Tuesday
-
 
+
-
</td>
+
</td>  
-
     <td><table id="intable" width="615" border="0" cellspacing="0" cellpadding="1">
+
     <td><table id="intable" width="615" border="0" cellspacing="0" cellpadding="1">  
    
    
       <td width="140"> •Cut: 13K+10I, 22M+10I
       <td width="140"> •Cut: 13K+10I, 22M+10I
-
</td>
+
</td>  
    
    
    
    
-
     <td width="181" > • Purify: 13K+10I, 22M+10I<br/>
+
     <td width="181" > • Purify: 13K+10I, 22M+10I<br/>  
• Ligation: Pvgb+22M+10I, Pnirb+13K+10I
• Ligation: Pvgb+22M+10I, Pnirb+13K+10I
-
</td>
+
</td>  
-
   <td width="142">• PCR backbones<br/>
+
   <td width="142">• PCR backbones<br/>  
• Electrophresis
• Electrophresis
-
</td>
+
</td>  
-
   <td width="144">• Gel excision and purification</td>
+
   <td width="144">• Gel excision and purification</td>  
-
</table></td>
+
</table></td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
     <td id="sheetleft">Aug.3rd
     <td id="sheetleft">Aug.3rd
Wednesday
Wednesday
-
 
+
-
</td>
+
</td>  
-
     <td><table width="640" border="0" cellspacing="0" cellpadding="1">
+
     <td><table width="640" border="0" cellspacing="0" cellpadding="1">  
-
   <tr>
+
   <tr>  
     <td width="170">• Make Phusion Buffer, 5×isothermel buffer
     <td width="170">• Make Phusion Buffer, 5×isothermel buffer
-
</td>
+
</td>  
     <td width="184">• colony PCR: 20H, 20J, 22B
     <td width="184">• colony PCR: 20H, 20J, 22B
-
</td>
+
</td>  
-
 
+
-
   </tr>
+
   </tr>  
-
</table>
+
</table>  
-
</td>
+
</td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
     <td id="sheetleft">Aug.4th
     <td id="sheetleft">Aug.4th
Thursday
Thursday
-
 
+
-
</td>
+
</td>  
-
     <td><table width="618" border="0" cellspacing="0" cellpadding="1">
+
     <td><table width="618" border="0" cellspacing="0" cellpadding="1">  
-
   <tr>
+
   <tr>  
     <td >• Cut: 10I+22M, 10I+13; and purification  
     <td >• Cut: 10I+22M, 10I+13; and purification  
-
</td>
+
</td>  
-
<td>• Ligation: Pvgb+22M+10I, Pnirb+13K+10I,<br/>
+
<td>• Ligation: Pvgb+22M+10I, Pnirb+13K+10I,<br/>  
• colony PCR: 13K+10I
• colony PCR: 13K+10I
-
</td>
+
</td>  
-
<td>• Culture: 20H, 20J, 22B, 1K, 1I,3C, 5E, 7C</td>
+
<td>• Culture: 20H, 20J, 22B, 1K, 1I,3C, 5E, 7C</td>  
-
 
+
-
<td>• Transform: 1G, 3A, 5A, 7A from the distribution plate</td>
+
<td>• Transform: 1G, 3A, 5A, 7A from the distribution plate</td>  
-
 
+
-
   </tr>
+
   </tr>  
-
</table>
+
</table>  
-
</td>
+
</td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
     <td id="sheetleft">Aug.5th
     <td id="sheetleft">Aug.5th
Friday
Friday
-
 
+
-
</td>
+
</td>  
-
     <td><table width="627" border="0" cellspacing="0" cellpadding="1">
+
     <td><table width="627" border="0" cellspacing="0" cellpadding="1">  
-
       <tr>
+
       <tr>  
-
       <td >• Check the plates. Contamination, or no positive colonies</td>
+
       <td >• Check the plates. Contamination, or no positive colonies</td>  
-
       <td>• Miniprep: 5 backbones, 22B-1, 22B-3</td>
+
       <td>• Miniprep: 5 backbones, 22B-1, 22B-3</td>  
-
       <td>• PCR: nirB from 13K+10I+nirB to firm the ligation. One positive result.</td>
+
       <td>• PCR: nirB from 13K+10I+nirB to firm the ligation. One positive result.</td>  
-
       <td>• Culture the positive colony.</td>
+
       <td>• Culture the positive colony.</td>  
-
       </tr>
+
       </tr>  
-
     </table></td>
+
     </table></td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
     <td id="sheetleft">Aug.6th
     <td id="sheetleft">Aug.6th
Saturday
Saturday
-
 
+
-
</td>
+
</td>  
-
     <td><table width="620" border="0" cellspacing="0" cellpadding="1">
+
     <td><table width="620" border="0" cellspacing="0" cellpadding="1">  
-
   <tr>
+
   <tr>  
      
      
-
 
+
-
   </tr>
+
   </tr>  
-
</table>
+
</table>  
-
</td>
+
</td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
     <td id="sheetleft">Aug.7th
     <td id="sheetleft">Aug.7th
Sunday
Sunday
-
</td>
+
</td>  
-
     <td><table width="620" border="0" cellspacing="0" cellpadding="1">
+
     <td><table width="620" border="0" cellspacing="0" cellpadding="1">  
-
       <tr>
+
       <tr>  
-
         <td width="244">• Cut: 22M,13K with E & S
+
         <td width="244">• Cut: 22M,13K with E &amp; S
-
 
+
-
</td>
+
</td>  
-
<td width="164">• Culture the red colonies from plate of pSB1C3</td>
+
<td width="164">• Culture the red colonies from plate of pSB1C3</td>  
-
<td>• PCR: 5 backbones<br/>
+
<td>• PCR: 5 backbones<br/>  
• Cut the PCR products with P+E and run the gel to confirm.
• Cut the PCR products with P+E and run the gel to confirm.
-
</td>
+
</td>  
-
       </tr>
+
       </tr>  
-
     </table></td>
+
     </table></td>  
-
   </tr>
+
   </tr>  
-
 
+
-
</table>
+
</table>  
-
 
+
-
</div>
+
</div>  
-
 
+
-
<div class="block" id="summ">
+
<div class="block" id="summ">  
-
<h3 style="color:white;">Data&protocol</h3>
+
<h3 style="color:white;">Data&amp;protocol</h3>  
-
<hr/>
+
<hr/>  
-
<div class="block" id="monday">
+
<div class="block" id="monday">  
-
<h3>Monday</h3>
+
<h3>Monday</h3>  
-
<hr/>
+
<hr/>  
-
<p>WEEK3</p>
+
<p>WEEK3</p>  
-
</div>
+
</div>  
-
<div class="block" id="tuesday">
+
<div class="block" id="tuesday">  
-
<h3>Tuesday</h3>
+
<h3>Tuesday</h3>  
-
<hr/>
+
<hr/>  
-
<p>bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb</p></div>
+
<p>bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb</p></div>  
-
<p>&nbsp;</p>
+
<p>&nbsp;</p>  
-
</div>
+
</div>  
-
</div>
+
</div>  
-
<!-- 55555555555555555555555555555555555555 -->
+
<!-- 55555555555555555555555555555555555555 -->  
-
<div class="inner" id="week6">
+
<div class="inner" id="week6">  
-
<div class="block" id="nsheet">
+
<div class="block" id="nsheet">  
-
<h3>Week6</h3><hr/>
+
<h3>Week6</h3><hr/>  
-
 
+
-
<table id="notesheet" width="650" border="1" cellspacing="0" cellpadding="1">
+
<table id="notesheet" width="650" border="1" cellspacing="0" cellpadding="1">  
-
   <tr><td width="76">Day</td><td width="349">Note</td></tr>
+
   <tr><td width="76">Day</td><td width="349">Note</td></tr>  
    
    
-
   <tr>
+
   <tr>  
     <td id="sheetleft">Aut.8th
     <td id="sheetleft">Aut.8th
Monday
Monday
-
 
+
-
</td>
+
</td>  
-
     <td><table id="intable" width="603" border="0" cellspacing="0" cellpadding="1">
+
     <td><table id="intable" width="603" border="0" cellspacing="0" cellpadding="1">  
    
    
     <td width="191">• The gel results of 5 backbones are right.
     <td width="191">• The gel results of 5 backbones are right.
-
</td>
+
</td>  
    
    
    
    
-
     <td width="249" >• Miniprep: pSB1C3, vgb+22M+10I</td><br />
+
     <td width="249" >• Miniprep: pSB1C3, vgb+22M+10I</td><br />  
-
<td width="157">• Culture vgb+22M+1oI in hypoxia<br/>
+
<td width="157">• Culture vgb+22M+1oI in hypoxia<br/>  
•PCR pSB1C3
•PCR pSB1C3
-
</td>
+
</td>  
    
    
-
</table></td>
+
</table></td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
     <td id="sheetleft">Aug.9th
     <td id="sheetleft">Aug.9th
Tuesday
Tuesday
-
 
+
-
 
+
-
</td>
+
</td>  
-
     <td><table id="intable" width="615" border="0" cellspacing="0" cellpadding="1">
+
     <td><table id="intable" width="615" border="0" cellspacing="0" cellpadding="1">  
    
    
       <td width="140"> • Run the PCR product
       <td width="140"> • Run the PCR product
-
</td>
+
</td>  
    
    
    
    
     <td width="181" > • PCR pSB1C3 again
     <td width="181" > • PCR pSB1C3 again
-
</td>
+
</td>  
   <td width="142">• Run the product, results are good.
   <td width="142">• Run the product, results are good.
-
</td>
+
</td>  
   
   
-
</table></td>
+
</table></td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
     <td id="sheetleft">Aug.10th
     <td id="sheetleft">Aug.10th
Wednesday
Wednesday
-
 
+
-
 
+
-
</td>
+
</td>  
-
     <td><table width="640" border="0" cellspacing="0" cellpadding="1">
+
     <td><table width="640" border="0" cellspacing="0" cellpadding="1">  
-
   <tr>
+
   <tr>  
-
     <td width="170">• Culture: 20H, 20J<br/>
+
     <td width="170">• Culture: 20H, 20J<br/>  
• Transform 13K from plate 3
• Transform 13K from plate 3
-
 
+
-
</td>
+
</td>  
     <td width="184">• Miniprep: 20H-1, 20J-1
     <td width="184">• Miniprep: 20H-1, 20J-1
-
</td>
+
</td>  
-
<td>• Cut: the 20H-1, 20J-1, 22B-1, 22B-3 with E+P</td>
+
<td>• Cut: the 20H-1, 20J-1, 22B-1, 22B-3 with E+P</td>  
-
   </tr>
+
   </tr>  
-
</table>
+
</table>  
-
</td>
+
</td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
     <td id="sheetleft">Aug.11th
     <td id="sheetleft">Aug.11th
Thursday
Thursday
-
 
+
-
 
+
-
</td>
+
</td>  
-
     <td><table width="618" border="0" cellspacing="0" cellpadding="1">
+
     <td><table width="618" border="0" cellspacing="0" cellpadding="1">  
-
   <tr>
+
   <tr>  
     <td >• Run the digestion results. Bands are confirmed right.
     <td >• Run the digestion results. Bands are confirmed right.
-
</td>
+
</td>  
<td>• Colony PCR vgb+22M+10I
<td>• Colony PCR vgb+22M+10I
-
</td>
+
</td>  
<td>• Cut the plasmid of vgb+22M+10I
<td>• Cut the plasmid of vgb+22M+10I
-
 
+
-
</td>
+
</td>  
-
 
+
-
 
+
-
   </tr>
+
   </tr>  
-
</table>
+
</table>  
-
</td>
+
</td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
     <td id="sheetleft">Aug.12th
     <td id="sheetleft">Aug.12th
Friday
Friday
-
 
+
-
 
+
-
</td>
+
</td>  
-
     <td><table width="627" border="0" cellspacing="0" cellpadding="1">
+
     <td><table width="627" border="0" cellspacing="0" cellpadding="1">  
-
       <tr>
+
       <tr>  
-
       <td >• Make new stock of competent cells</td>
+
       <td >• Make new stock of competent cells</td>  
-
       <td>• Ligation: 13K+10I<br/>
+
       <td>• Ligation: 13K+10I<br/>  
• Cut the PCR results and 22M with E+P
• Cut the PCR results and 22M with E+P
-
</td>
+
</td>  
-
       <td>• Sequence: nirB, vgbL, 12I, 20H, 20J, 22B</td>
+
       <td>• Sequence: nirB, vgbL, 12I, 20H, 20J, 22B</td>  
-
       <td>• Transform 13K+10I+pSB1K3<br/>
+
       <td>• Transform 13K+10I+pSB1K3<br/>  
• Cut 13K to validate. Results are right.
• Cut 13K to validate. Results are right.
-
</td>
+
</td>  
-
       </tr>
+
       </tr>  
-
     </table></td>
+
     </table></td>  
-
   </tr>
+
   </tr>  
-
   <tr>
+
   <tr>  
     <td id="sheetleft">Aug.13th
     <td id="sheetleft">Aug.13th
-
Saturday
 
-
 
+
Saturday
-
</td>
+
-
     <td><table width="620" border="0" cellspacing="0" cellpadding="1">
+
-
   <tr>
+
</td>  
-
     <td>• Colony PCR: 13K+10I+pSB1K3</td>
+
     <td><table width="620" border="0" cellspacing="0" cellpadding="1">  
-
     <td>• Culture: vgb+22M+10I in 5ml(shaking), 5ml(water bath), 10ml(water bath), 15(water bath).</td>
+
   <tr>  
-
     <td>• Check the YFP expression of 5ml(shaking) and 15ml(water bath). No yellow fluorescent cells,</td>
+
     <td>• Colony PCR: 13K+10I+pSB1K3</td>  
-
 
+
     <td>• Culture: vgb+22M+10I in 5ml(shaking), 5ml(water bath), 10ml(water bath), 15(water bath).</td>  
-
   </tr>
+
     <td>• Check the YFP expression of 5ml(shaking) and 15ml(water bath). No yellow fluorescent cells,</td>  
-
</table>
+
-
</td>
+
   </tr>  
-
   </tr>
+
</table>  
-
   <tr>
+
</td>  
 +
   </tr>  
 +
   <tr>  
     <td id="sheetleft">Aug.14th
     <td id="sheetleft">Aug.14th
Sunday
Sunday
-
 
+
-
</td>
+
</td>  
-
     <td><table width="620" border="0" cellspacing="0" cellpadding="1">
+
     <td><table width="620" border="0" cellspacing="0" cellpadding="1">  
-
       <tr>
+
       <tr>  
         <td width="244">• Transform: the  Gibson assembly results.
         <td width="244">• Transform: the  Gibson assembly results.
-
 
+
-
</td>
+
</td>  
-
<td width="164">• Miniprep: 13K+10I<br/>
+
<td width="164">• Miniprep: 13K+10I<br/>  
• Cut: 13K+10I
• Cut: 13K+10I
-
</td>
+
</td>  
-
<td>• Purification: the digestion results.<br/>
+
<td>• Purification: the digestion results.<br/>  
• Ligation: nirB+13K+10I
• Ligation: nirB+13K+10I
-
 
+
-
</td>
+
</td>  
-
       </tr>
+
       </tr>  
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     </table></td>  
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+
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-
 
+
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+
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-
 
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<div class="block" id="summ">  
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+
<h3 style="color:white;">Data&amp;protocol</h3>  
-
<hr/>
+
<hr/>  
-
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-
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-
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-
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+
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Revision as of 15:51, 26 September 2011

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">

Week1

DayNote
Jul.4th Monday
• Aerobic cultivation of DH5α • Preparation of apparatus for the formation of biofilm
Jul 5th Tuesday  
Jul.6th Wednesday
•Receiving primers ordered previously
Jul.7th Thursday
• Preparation of the aliquot of the primers • Something wrong with a shaking incubator
Jul.8th Friday
• Preparation of culture plates for the transformations
Jul.9th Saturday
• Preparation of culture plates for the transformations • protocols of transformation
Jul.10th Sunday
• Several colonies were picked up and cultivated in 5mL LB medium. •Cryosectioning of biofilm

 

Data&protocol

Monday

blanknote

There is no data in first week

 

Week2

DayNote
Jul.11th Monday
• Set up new LB culture plates with ampicillin and kanamycin

 • ·Sterilization of Glycerol and
·Preparation of 25mg/mL kanamycin		 •Transformation of the parts mentioned on Jul.9th for the second time •Observation the sections
Jul 12th Tuesday
• Pick two colonies of each parts and cultivate them in LB medium •Transformation of three parts(20J,20H.22B from Kit plates of 2011 Distribution ) which are related to the degradation of Cellulose •·Mini prep to isolate 10I,12I and 22M •Conservation of 10I,12I,22M and 11P
Jul.13th Wednesday
• Place the culture plate of 20J,20H and 22B in the fridge. •Min prep to isolate 13K •Conservation of 13K •Restriction digest of the parts(12I,10I,22M,13K) with EcoRI and PstI •Gel electrophoresis to analyse restriction fragments •Test the Tm of Primers CP1&CS,NP&NS with 13K. The result of Gel electrophoresis shows that 60.2℃ is the Tm of NS and NP, and 57.4℃ is the Tm of CS and CP1.
Jul.14th Thursday
• Test the Tm of the YFP,RFP,tetR,Vgb and nirB primers. The result of Gel electrophoresis shows that the Tm for the primers of Vgb is 54℃ and the Tm for the primers of nirB is 55℃.The RCR of YFP,RFP and tetR failed.
Jul.15th Friday
• PCR(Phusion)
• Digest 10I, 22M, 13K • Used wrong cutter, digestion again. 		• Run the results of PCR and the first digestion. The annealing temperature of YFP needed change. The digestion results confirmed • Run the digestion results of second time. The bands are confirmed.
Jul.16th Saturday
• Cut the linearized pSB1k3 with E+P • Purify the digestion results of 22M, 13K, 10I • Confirm digestion of pSB1k3 by electrophoresis, then purification • Test Tm of YFP
• ligation: 22M+10I, 13K+10I 		• Tm of YFP is 54 degree • transform the ligation results.
Jul.17th Sunday
• PCR 22M
• purify Vgb, RFP, RFP-tetR, YFP-tetR. Gibson PCR. 		• ligation the purified the fragments in yesterday. • 22M PCR
• Transform the ligation results.

 

Data&protocol

Jul.13th Wednesday

Systems of restriction digestion with EcoRI and PstI

Plasmid 1μL (>100ng)
EcoRI 1μL
PstI 1μL
10×Buffer Tango 2μL
ddH2O 15μL

Temperature grad
56℃,57.4℃,60.2℃,62.9℃,64.3℃,65.8℃

 

PCR system (test the Tm of the primers CP1&CS, NP&NS)

10×Buffer 2μL
dNTPs 0.5μL
primers CP1 (NP) 1μL
primers CS (NS) 1μL
Template 1μL
rTaq 0.2μL
H2O 14.5μL
Total 20μL

Jul.14th Thursday

PCR systems(test the Tm of the primers for YFP,RFP,tetR,Vgb and nirB )

10×Buffer 2μL
dNTPs 0.5μL
primers F 0.5μL
primers R 0.5μL
Template 1μL
Taq 0.2μL
H2O 15.5μL
Total 20μL

 

Temperature grad and the results:

vgb&nirB pcr test:

 

 

Week3

DayNote
Jul.18th Monday
• Genome extraction of E.coli • PCR of NirB
• cut 10I with X+P, cut 22M, 13K with E+S, cut pSB1k3 with E+P		 • Ligate: 10I+13K, 10I+22M
• Repeat NirB PCR

• Culture 11P
• Miniprep 10I, 22M, 13K
Jul 19th Tuesday
• mini-prep: 10I+13K, 10I+22M •insert 10I+13K, 10I+22M into pSB1k3 •transform: 5E, 3C, 7C, 1K, 1I from the distribution plate •transform 10I+22M, 10I+13K
Jul.20th Wednesday
•Colonies of 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I confirmed • Gibson PCR •Colony PCR 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I •PCR NirB
Jul.21th Thursday
•PCR verification of 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I • Gibson PCR • Run the results of PCR verification. Bands confirmed. • Culture the 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I • Purification of Gibson PCR results
Jul.22th Friday
• PCR amplification of Gibson assembly results • Mini prep: 22M+10I, 22.Presever in -20
• Medi prep: 1K,1I,3C,5E,7C
• Gibson Assembly fail. 		• PCR NirB by Phusion
•Repeat PCR by changing Pnibr to Gnirbr
•Cut the mini and medi prep results with E 		•Run the results of PCR and digestion. Fail in PCR of NirB, succeed in 10I+3K and 10I+22M ligation.
Jul.23th Saturday
•Double digestion of 13K+10I, 22M+10I, pSB1c3 • Fail in purification of Medi prep • PCR NirB
• Ligate NirB+13K+10I, Vgb+22M+10I
Jul.24th Sunday
• Gibson assembly: NirB+RFP+tetR
• Cut results of Gibson assembly and pSB1c3. 		• Purify the ligation results in yesterday • ligate with backbone • Culture 1I, 1K, 3C, 5E, 7C, 11P

 

Data&protocol

Monday

WEEK3

Tuesday

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Week4

DayNote
Jul.25th Monday
• Transform Pnirb+13k+10I+pSBK3-2 • Place the culture in 4 degree • Medi-and mini-prep of five cultre •Gibson Assembly confirmation by gel
Jul. 26th Tuesday
• Cut Pnirb, Pvgb,(E+S)
• Cut 10I+13K, 10I+22M.(X+P) 		• Ligation: vgb+10I+22M, nirB+10I+13K, • Ligation: pSBK13+vgb+10I+22M, pSBK13+nirB+10I+13K • Transform the ligation results.
Jul.27th Wednesday
• No colony on the plate
• Gibson assembly 		• Run the results of Gibson assembly • Excise the bands, the purify the DNA
• Gibson Assenmly
Jul.28th Thursday
•2011 China Meet-up @ Hefei
Jul.29th Friday
•2011 China Meet-up @ Hefei
Jul.30th Saturday
Jul.31th Sunday
• Culture 22M+10I, 13K+10I • Cut a0H, 20J, 22B

 

Data&protocol

Monday

WEEK3

Tuesday

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Week5

DayNote
Aug.1st Monday
•Fusion PCR Vgb+YFP+tetR, NirB+RFP+tetR, •PCR: NirB, Vgb
Aug.2nd Tuesday
•Cut: 13K+10I, 22M+10I • Purify: 13K+10I, 22M+10I
• Ligation: Pvgb+22M+10I, Pnirb+13K+10I 		• PCR backbones
• Electrophresis 		• Gel excision and purification
Aug.3rd Wednesday
• Make Phusion Buffer, 5×isothermel buffer • colony PCR: 20H, 20J, 22B
Aug.4th Thursday
• Cut: 10I+22M, 10I+13; and purification • Ligation: Pvgb+22M+10I, Pnirb+13K+10I,
• colony PCR: 13K+10I 		• Culture: 20H, 20J, 22B, 1K, 1I,3C, 5E, 7C • Transform: 1G, 3A, 5A, 7A from the distribution plate
Aug.5th Friday
• Check the plates. Contamination, or no positive colonies • Miniprep: 5 backbones, 22B-1, 22B-3 • PCR: nirB from 13K+10I+nirB to firm the ligation. One positive result. • Culture the positive colony.
Aug.6th Saturday
Aug.7th Sunday
• Cut: 22M,13K with E & S • Culture the red colonies from plate of pSB1C3 • PCR: 5 backbones
• Cut the PCR products with P+E and run the gel to confirm.

Data&protocol

Monday

WEEK3

Tuesday

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Week6

DayNote
Aut.8th Monday
• The gel results of 5 backbones are right. • Miniprep: pSB1C3, vgb+22M+10I • Culture vgb+22M+1oI in hypoxia
•PCR pSB1C3
Aug.9th Tuesday
• Run the PCR product • PCR pSB1C3 again • Run the product, results are good.
Aug.10th Wednesday
• Culture: 20H, 20J
• Transform 13K from plate 3 		• Miniprep: 20H-1, 20J-1 • Cut: the 20H-1, 20J-1, 22B-1, 22B-3 with E+P
Aug.11th Thursday
• Run the digestion results. Bands are confirmed right. • Colony PCR vgb+22M+10I • Cut the plasmid of vgb+22M+10I
Aug.12th Friday
• Make new stock of competent cells • Ligation: 13K+10I
• Cut the PCR results and 22M with E+P 		• Sequence: nirB, vgbL, 12I, 20H, 20J, 22B • Transform 13K+10I+pSB1K3
• Cut 13K to validate. Results are right.
Aug.13th Saturday
• Colony PCR: 13K+10I+pSB1K3 • Culture: vgb+22M+10I in 5ml(shaking), 5ml(water bath), 10ml(water bath), 15(water bath). • Check the YFP expression of 5ml(shaking) and 15ml(water bath). No yellow fluorescent cells,
Aug.14th Sunday
• Transform: the Gibson assembly results. • Miniprep: 13K+10I
• Cut: 13K+10I 		• Purification: the digestion results.
• Ligation: nirB+13K+10I

Data&protocol

Monday

WEEK3

Tuesday

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Data&protocol

Monday

WEEK7

Tuesday

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Data&protocol

Monday

WEEK8

Tuesday

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Data&protocol

Monday

WEEK9

Tuesday

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Data&protocol

Monday

WEEK10

Tuesday

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Data&protocol

Monday

WEEK11

Tuesday

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Data&protocol

Monday

WEEK12

Tuesday

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Data&protocol

Monday

WEEK13

Tuesday

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Retrieved from "http://2011.igem.org/Team:ZJU-China/Notebook"