Difference between revisions of "Team:Aix-Marseille/Notebook"

 
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color: #FFFFFF !important;
 
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} .bg-1 {
 
} .bg-1 {
background: url(http://i.imgur.com/FrsJhjG.jpg);background-repeat:no-repeat;
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background: url(https://static.igem.org/mediawiki/2015/f/f3/FrsJhjG.jpg);background-repeat:no-repeat;
 
         background-size:cover;
 
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min-height: 600px;
 
min-height: 600px;
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               </script>
 
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<body>
 
<body>
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                         </button>
 
                         </button>
                         <a href="/Team:Aix Marseille" class="navbar-brand hidden-lg hidden-md hidden-sm" ><http://i.imgur.com/oHSATfM.png" class="img-responsive" width="80" height="80"></a>
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                             <div class="collapse navbar-collapse  " id="bs-example-navbar-collapse-1">
 
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                                 <li><a href="/Team:Aix-Marseille"  class="logo"><img src="http://i.imgur.com/oHSATfM.png" class="img-responsive" width="85" height="85"></a></li>
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                                 <li><a href="/Team:Aix-Marseille"  class="logo"><img src="https://static.igem.org/mediawiki/2015/6/6e/OHSATfM.png" class="img-responsive" width="85" height="85"></a></li>
 
                                     <li  ><a href="/Team:Aix-Marseille">Home</a></li>
 
                                     <li  ><a href="/Team:Aix-Marseille">Home</a></li>
 
                                       <li class="dropdown " id="dropdown"><a href="#" class="dropdown-toggle"  data-toggle="dropdown">Project</a>
 
                                       <li class="dropdown " id="dropdown"><a href="#" class="dropdown-toggle"  data-toggle="dropdown">Project</a>
 
                                       <ul class="dropdown-menu">
 
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                                         <li ><a href="/Team:Aix-Marseille/Description">Description</a></li>
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                                         <li ><a href="/Team:Aix-Marseille/Project/Description">Overview</a></li>
 
                                         <li ><a href="/Team:Aix-Marseille/Design">Project design</a></li>
 
                                         <li ><a href="/Team:Aix-Marseille/Design">Project design</a></li>
 
                                         <li ><a href="/Team:Aix-Marseille/Parts">Parts</a></li>                                   
 
                                         <li ><a href="/Team:Aix-Marseille/Parts">Parts</a></li>                                   
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                                     <li class="dropdown " id="dropdown"><a href="#" class="dropdown-toggle" data-toggle="dropdown">Interlab Study</a>
 
                                     <li class="dropdown " id="dropdown"><a href="#" class="dropdown-toggle" data-toggle="dropdown">Interlab Study</a>
 
                                       <ul class="dropdown-menu">
 
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                                        <li ><a href="/Team:Aix-Marseille/Notebook2">Calendar</a></li>
 
                                         <li ><a href="/Team:Aix-Marseille/Measurement">Results</a></li>
 
                                         <li ><a href="/Team:Aix-Marseille/Measurement">Results</a></li>
                                         <li ><a href="/Team:Aix-Marseille/Protocols2">Protocols2</a></li>
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                                         <li ><a href="/Team:Aix-Marseille/Protocols2">Protocols</a></li>
 
                                         <li ><a href="/Team:Aix-Marseille/Sequencing/data">Sequencing data</a></li>                                   
 
                                         <li ><a href="/Team:Aix-Marseille/Sequencing/data">Sequencing data</a></li>                                   
 
                                       </ul>
 
                                       </ul>
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                                       </ul>     
 
                                       </ul>     
 
                                     </li>
 
                                     </li>
                        
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                       <li class="dropdown " id="dropdown"><a href="/Team:Aix-Marseille/Achievement" class="dropdown-toggle"  data-toggle="dropdown">Achievement</a></li>
 
                                   </li>
 
                                   </li>
  
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     </header>
 
     </header>
 
 
 
 
  
  
  
 
<div class="container">
 
<div class="container">
   <center>{<h2>Calendar</h2>}
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   <h2 class="title wow bounceInUp"><center><span style ="color:#8E3B8C"><span style="font-family:Armalite rifle">CALENDAR</center></span></h2></span>
  
  
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   <button type="button" class="btn btn-info" data-toggle="collapse" style="width:110px;" data-target="#demo5">April 12</button>
 
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width:110px;" data-target="#demo5">April 12</button>
 
   <div id="demo5" class="collapse">
 
   <div id="demo5" class="collapse">
Presentation of all subject ideas. Election of the best, called “Chew Fight”. We decided to work on the chew degradation using enzymes produced by E.Coli.
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Presentation of all subject ideas. Election of the best, called “Chew Fight”. We decided to work on the chew degradation using enzymes produced by <i>E.Coli</i>.
 
</div>
 
</div>
 
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo6">April 19</button>
 
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo6">April 19</button>
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   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo9">April 23</button>
 
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo9">April 23</button>
 
   <div id="demo9" class="collapse">
 
   <div id="demo9" class="collapse">
https://www.facebook.com/photo.php?fbid=10206323650749123&set=gm.803648703058628&type=1
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<img src="https://static.igem.org/mediawiki/2015/0/01/Sac.png">
 
A present from the scientific culture unit of the AMU: a new backpack for each team member: we are ready to come to Boston !
 
A present from the scientific culture unit of the AMU: a new backpack for each team member: we are ready to come to Boston !
 
   </div>
 
   </div>
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   <div id="demo10" class="collapse">
 
   <div id="demo10" class="collapse">
 
Creation of the logo of the AMU team :
 
Creation of the logo of the AMU team :
https://www.facebook.com/photo.php?fbid=10206406746426463&set=gm.809706195786212&type=1
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<img src="https://static.igem.org/mediawiki/2015/4/4d/Logo1.png">
 
   </div>
 
   </div>
 
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo11">May 11</button>
 
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo11">May 11</button>
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   <div id="demo18" class="collapse">
 
   <div id="demo18" class="collapse">
 
The linker between the laccase and the cytochrome has been designed by Wilson.
 
The linker between the laccase and the cytochrome has been designed by Wilson.
https://www.facebook.com/photo.php?fbid=10207511567166932&set=gm.830198770403621&type=1
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<img src="https://static.igem.org/mediawiki/2015/8/83/Firstmodeling.jpeg">
 
The sequences needed were ordered (IDT).
 
The sequences needed were ordered (IDT).
 
   </div>
 
   </div>
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First day at the laboratory, meeting of everyone.
 
First day at the laboratory, meeting of everyone.
 
Cleaning and room arrangement.
 
Cleaning and room arrangement.
https://www.facebook.com/photo.php?fbid=102sa06853955166402&set=gm.834872003269631&type=1
 
 
   </div>
 
   </div>
 
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo21">June 19</button>
 
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo21">June 19</button>
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   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo25">June 25</button>
 
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo25">June 25</button>
 
   <div id="demo25" class="collapse">
 
   <div id="demo25" class="collapse">
<p>Resuspension of biobricks K863011, K863006, K525998, K844000, E0240, K823005, K823012, I20260 and transformation into E.coli (DH5-alpha). </p>
+
<p>Resuspension of biobricks K863011, K863006, K525998, K844000, E0240, K823005, K823012, I20260 and transformation into <i>E.coli</i> (DH5-alpha). </p>
 
<p>Negative results (no colony on the plates).</p>
 
<p>Negative results (no colony on the plates).</p>
 
   </div>
 
   </div>
 
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo26">June 26</button>
 
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo26">June 26</button>
 
   <div id="demo26" class="collapse">
 
   <div id="demo26" class="collapse">
<p>New try for Resuspension of biobricks K863011, K863006, K525998, K844000, E0240, K823005, K823012, I20260 and transformation into E.coli (DH5-alpha). </p>
+
<p>New try for Resuspension of biobricks K863011, K863006, K525998, K844000, E0240, K823005, K823012, I20260 and transformation into <i>E.coli</i> (DH5-alpha). </p>
 
<p>Positive results (several colonies on the plate).</p>
 
<p>Positive results (several colonies on the plate).</p>
 
   </div>
 
   </div>
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<p>Colony PCR on BL21 strain to amplify the 3 parts of ccm operon. Oligos were too concentrated and inhibited the reaction.</p>
 
<p>Colony PCR on BL21 strain to amplify the 3 parts of ccm operon. Oligos were too concentrated and inhibited the reaction.</p>
 
<p>Colony PCR and electrophoresis to check the size of the biobrick “12” (cytochrome c Shewanella). Positive result.</p>
 
<p>Colony PCR and electrophoresis to check the size of the biobrick “12” (cytochrome c Shewanella). Positive result.</p>
<p>Resuspension of biobricks “05”, “08”, “30”, “11” received from IDT. E/P digestion, ligation into pSB1C3, transformation into E.coli (TG1). Negative result (no colony on the plate).</p>
+
<p>Resuspension of biobricks “05”, “08”, “30”, “11” received from IDT. E/P digestion, ligation into pSB1C3, transformation into <i>E.coli</i> (TG1). Negative result (no colony on the plate).</p>
 
   </div>
 
   </div>
 
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width:110px;" data-target="#demo35">July 16</button>
 
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width:110px;" data-target="#demo35">July 16</button>
 
   <div id="demo35" class="collapse">
 
   <div id="demo35" class="collapse">
<p>Second try to resuspend the biobricks “05”, “08”, “30”, “11” received from IDT and resuspension of the biobrick “13” received from IDT. E/P digestion, ligation into pSB1C3, transformation into E.Coli (TG1). Positive result (several colonies on the plate).</p>
+
<p>Second try to resuspend the biobricks “05”, “08”, “30”, “11” received from IDT and resuspension of the biobrick “13” received from IDT. E/P digestion, ligation into pSB1C3, transformation into <i>E.Coli</i> (TG1). Positive result (several colonies on the plate).</p>
 
<p>Second try for Colony PCR on DH5-alpha strain to amplify the 3 parts of ccm operon with diluted oligos at 1/10. Positive result except for part 1: there are some contaminants due to nonspecific hybridization.</p>
 
<p>Second try for Colony PCR on DH5-alpha strain to amplify the 3 parts of ccm operon with diluted oligos at 1/10. Positive result except for part 1: there are some contaminants due to nonspecific hybridization.</p>
<p>Reception of oligos from SIGMA to amplify the laccase of E.coli and T.thermophilus.</p>
+
<p>Reception of oligos from SIGMA to amplify the laccase of <i>E.coli</i> and T.thermophilus.</p>
 
   </div>
 
   </div>
 
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo36">July 17</button>
 
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo36">July 17</button>
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<p>Second try for high fidelity DNA amplification of biobricks “09” and “10” by PCR with the Q5 polymerase in order to remove the codon-STOP (the new synthesized biobricks are called respectively “35” and “36”). PCR clean up. Negative result (non-specific amplification). An electrophoresis was done on the total PCR product and was extracted and purified by PCR clean up.</p>
 
<p>Second try for high fidelity DNA amplification of biobricks “09” and “10” by PCR with the Q5 polymerase in order to remove the codon-STOP (the new synthesized biobricks are called respectively “35” and “36”). PCR clean up. Negative result (non-specific amplification). An electrophoresis was done on the total PCR product and was extracted and purified by PCR clean up.</p>
 
<p>Miniprep of “08”. E/P digestion to check the size of the insert. Negative result (unexpected size).</p>
 
<p>Miniprep of “08”. E/P digestion to check the size of the insert. Negative result (unexpected size).</p>
<p>Resuspension of biobrick “15” received from IDT. E/P digestion, ligation into pSB1C3, transformation into E.coli (DH5-alpha). Negative result (no colony on the plate).
+
<p>Resuspension of biobrick “15” received from IDT. E/P digestion, ligation into pSB1C3, transformation into <i>E.coli</i> (DH5-alpha). Negative result (no colony on the plate).
 
<p>New TG1 competent cells done.</p>
 
<p>New TG1 competent cells done.</p>
 
<p>Transformation efficiency test with the transformation efficiency kit of iGEM on the new DH5-alpha competent cells. Positive result (the DH5-alpha cells are competent enough to be transformed with our constructions).</p>
 
<p>Transformation efficiency test with the transformation efficiency kit of iGEM on the new DH5-alpha competent cells. Positive result (the DH5-alpha cells are competent enough to be transformed with our constructions).</p>
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   <div id="demo39" class="collapse">
 
   <div id="demo39" class="collapse">
 
<p>Colony PCR and electrophoresis to check the size of the biobricks “05” and “13”. Positive result. Starters of « 05 » and « 13 ».</p>
 
<p>Colony PCR and electrophoresis to check the size of the biobricks “05” and “13”. Positive result. Starters of « 05 » and « 13 ».</p>
<p>SLIC reaction to ligate the 3 parts of ccm operon into the pSB1C3 plasmid. Transformation into E.coli (TG1). Negative result (no colony on the plate). </p>
+
<p>SLIC reaction to ligate the 3 parts of ccm operon into the pSB1C3 plasmid. Transformation into <i>E.coli</i> (TG1). Negative result (no colony on the plate). </p>
<p>SLIC reaction to ligate the new biobricks “35” and “36” into pSB1C3. Transformation into E.coli (TG1). Negative result (no colony on the plate).</p>
+
<p>SLIC reaction to ligate the new biobricks “35” and “36” into pSB1C3. Transformation into <i>E.coli</i> (TG1). Negative result (no colony on the plate).</p>
 
<p>Transformation efficiency test with the transformation efficiency kit of iGEM on the new TG1 competent cells. Positive result (the TG1 cells are competent enough to be transformed with our constructions)</p>
 
<p>Transformation efficiency test with the transformation efficiency kit of iGEM on the new TG1 competent cells. Positive result (the TG1 cells are competent enough to be transformed with our constructions)</p>
 
   </div>
 
   </div>
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   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo41">July 24</button>
 
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo41">July 24</button>
 
   <div id="demo41" class="collapse">
 
   <div id="demo41" class="collapse">
<p>New try with different volumes for resuspension of biobricks “15” and “08” received from IDT. E/P digestion, ligation into pSB1C3, transformation into E.coli (TG1). Positive result (several colonies on the plate).</p>
+
<p>New try with different volumes for resuspension of biobricks “15” and “08” received from IDT. E/P digestion, ligation into pSB1C3, transformation into <i>E.coli</i> (TG1). Positive result (several colonies on the plate).</p>
 
<p>Colony PCR and electrophoresis to check the size of the biobricks”08”, “09”, “10”,”12-02”, “30-02”, “32” and “34”. Positive result for “09” and “10” (expected size). Starters of « 09 » and « 10 ».</p>  
 
<p>Colony PCR and electrophoresis to check the size of the biobricks”08”, “09”, “10”,”12-02”, “30-02”, “32” and “34”. Positive result for “09” and “10” (expected size). Starters of « 09 » and « 10 ».</p>  
 
<p>Negative result for “32”, “34”, “12-02”, “30-02” (No colony on the plate) and for “08” (unexpected size).<
 
<p>Negative result for “32”, “34”, “12-02”, “30-02” (No colony on the plate) and for “08” (unexpected size).<
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<p>Plasmid purification of “13-02” “09-02” “10-02”</p>
 
<p>Plasmid purification of “13-02” “09-02” “10-02”</p>
 
<p>E/P digestion of “05” and “11” and ligation “05” + pSB1K3 and “11” + pSB1K3 </p>
 
<p>E/P digestion of “05” and “11” and ligation “05” + pSB1K3 and “11” + pSB1K3 </p>
<p>Transformation of ligation products into E.coli (TG1)</p>
+
<p>Transformation of ligation products into <i>E.coli</i> (TG1)</p>
 
<p>PCR on “07” and “08” => seems to be unclean with the Q5 high fidelity DNA amplification</p>
 
<p>PCR on “07” and “08” => seems to be unclean with the Q5 high fidelity DNA amplification</p>
 
<p>X/P digestion of “15” </p>
 
<p>X/P digestion of “15” </p>
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   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo50">August 5</button>
 
   <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo50">August 5</button>
 
   <div id="demo50" class="collapse">
 
   <div id="demo50" class="collapse">
<p>Transformation of ”01-15” and “12-02” into E.coli (TG1)</p>
+
<p>Transformation of ”01-15” and “12-02” into <i>E.coli</i> (TG1)</p>
 
<p>Plasmid purification of “09-02” “07” “13-02” “09-02” “10-02” => low concentration of “09-02” but we got good result for “13-02”</p>
 
<p>Plasmid purification of “09-02” “07” “13-02” “09-02” “10-02” => low concentration of “09-02” but we got good result for “13-02”</p>
 
<p>Transformation of “12-02” and “01-15” into TG1</p>
 
<p>Transformation of “12-02” and “01-15” into TG1</p>
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<p>Colony PCR of “05” and “01-15” --> no results on this PCR </p>
 
<p>Colony PCR of “05” and “01-15” --> no results on this PCR </p>
  
<p>Transformation of “09-02” “10-02” “01-15” into E.coli (TG1)</p>
+
<p>Transformation of “09-02” “10-02” “01-15” into <i>E.coli</i> (TG1)</p>
  
 
<p>Ligation “05” + pSB1K3 and “11” + pSB1K3</p>
 
<p>Ligation “05” + pSB1K3 and “11” + pSB1K3</p>
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<p>SDS-PAGE of “01-3502” “01-1202”</p>
 
<p>SDS-PAGE of “01-3502” “01-1202”</p>
  
<p>01-3502 purified and pure!!!! We got our first protein which is a laccase from E.coli</p>
+
<p>01-3502 purified and pure!!!! We got our first protein which is a laccase from <i>E.coli</i></p>
  
 
<p>Starters launched of “38” “13-02” “01-36” “01-15” “01-3502” “01-1202” “37” “35-02” “12-02” “35” “36” “01-35” “39” “40” “45” “48” </p>
 
<p>Starters launched of “38” “13-02” “01-36” “01-15” “01-3502” “01-1202” “37” “35-02” “12-02” “35” “36” “01-35” “39” “40” “45” “48” </p>
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<p>Colony PCR of “47” “01-3002” “43” “44” => almost everything is positive!!! Great day!</p>
 
<p>Colony PCR of “47” “01-3002” “43” “44” => almost everything is positive!!! Great day!</p>
 +
  </div>
 +
 +
<button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo75">September 7</button>
 +
 +
  <div id="demo75" class="collapse">
 +
<p>Plasmid purification (miniprep) of “44” “47” “43” “38” “01-1302” “01-1202” “01-3002” “45” </p>
 +
 +
<p>“01-1302” “01-36” “3813-02” seems good so they are send for sequencing.</p>
 +
 +
<p>Construction : Digestion E/S of “38” “01-36” “01-35” “01” and Digestion X/P of “44” “47” “43” “48” “44” “45” “1302” “3813-02” “39-02” “40” “3812-02”</p>
 +
 +
<p>Starter launched on “01-1202” “01-3002” for production and on “0136” for miniprep</p>
 +
 +
  </div>
 +
<button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo76">September 8</button>
 +
  <div id="demo76" class="collapse">
 +
<p>Ligation in pSB1C3 of “0136-02” “01-381302” “01-381202” “01-3902” “01-1302” “38-3902” “0136-40” “0136-45” “0136-48” “0135-43” “0135-44” “0135-47”</>
 +
 +
<p>Transformation of “01-3002” in BL21, of pSB4K5 and pSB3T5 in TG1 and transformation of ligation product in TG1</p>
 +
 +
<p>Purification of “01-1202” (1) and (2):</p>
 +
    <p>Induction by IPTG </p>
 +
    <p>Storing the cells at -80°C </p>
 +
  </div>
 +
 +
<button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo77">September 9</button>
 +
  <div id="demo77" class="collapse">
 +
<p>Colony PCR of “013002” “013002” => all results are positive</p>
 +
 +
<p>Production of “01-1202” : Sonication of cells, centrifugation 10 min at 4500 rpm, ultracentrifugation 45 min at 4500 rpm, solubilizing membrane in Triton 1% during 2h and incubation overnight.</p>
 +
  </div>
 +
<button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo78">September 10</button>
 +
  <div id="demo78" class="collapse">
 +
<p>Preparation of SDS Page 15% and a western blot with products of yesterday => results are not good so we have to resart the production</p>
 +
  </div>
 +
<button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo79">September 14</button>
 +
  <div id="demo79" class="collapse">
 +
<p>Culture of “01-1202” and “01-3002” in anaerobic condition, cells were centrifuged 10 min at 4500 rpm and pellet were stored at -20°C</p>
 +
<p>A western blot was done → Production of Cytochrome, monomer in the membrane and dimer in the cytoplasm</p>
 +
 +
<p>The next step is to purify “01-1202” and “01-3002” and start a culture on 01-3502 in anaerobic condition</p>
 
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Latest revision as of 00:06, 19 September 2015

Chew fight

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  • Aix Marseille Université
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Chew figth project, for the iGEM competition. See you soon in Boston !