Team:Toulouse/Notebook/Protocols

From 2014.igem.org

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   <div id="innercontenthome">
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       <div class="centering" style="padding-top: 85px; padding-bottom:40px;">
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  <div id="column-left">
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<h3 class="title2" style="margin-top:10px; color:#333;">Summary :</h3>
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<ul class="menuleft">
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  <li style="margin-top:25px;"><a href="#select1"><i>E. coli</i> competent cells</a></li>
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  <li><a href="#select2"><i>E. coli</i> transformation protocol</a></li>
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  <li><a href="#select3">Miniprep and alcaline lysis</a></li>
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  <li><a href="#select4">Cloning</a></li>
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  <li><a href="#select5">Checking of the genetic constructions</a></li>
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  <li><a href="#select6"><i>B. subtilis</i> transformation</a></li>
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  <li><a href="#select7">Checking of the genetic constructions after plasmid integration in <i>Bacillus subtilis</i> </a></li>
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<li><a href="#select8">Final Tests</a></li>
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  </ul>
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</div>
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<div class="column-right" style="width:75%; float:right;">
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<p class="title1"><I>E. coli</I> competent cells</p>
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<p class="title1" id="select1"><I>E. coli</I> competent cells</p>
<p class="texte"><I> <CENTER> MANIPULATION IN ICE  </CENTER> </I></p>
<p class="texte"><I> <CENTER> MANIPULATION IN ICE  </CENTER> </I></p>
<p class="texte"><B> Day 0 </B>
<p class="texte"><B> Day 0 </B>
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</p>
</p>
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<p class="title1"> <I>E. coli</I> transformation protocol </p>
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<p class="title1" id="select2"> <I>E. coli</I> transformation protocol </p>
<p class="texte">
<p class="texte">
- Let the LB agar medium plates dry in a sterile area
- Let the LB agar medium plates dry in a sterile area
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  </p>
  </p>
   
   
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<p class="title1"> Miniprep and alcaline lysis </p>
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<p class="title1" id="select3"> Miniprep and alcaline lysis </p>
<p class="texte"><B> Day 0 </B></p>
<p class="texte"><B> Day 0 </B></p>
<p class="texte">
<p class="texte">
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</I>
</I>
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<p class="title1"> Cloning </p>
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<p class="title1" id="select4"> Cloning </p>
<p class="texte">
<p class="texte">
<br>After taking the competent cells, transforming the Biobricks and making the miniprep, make the digestion mix.
<br>After taking the competent cells, transforming the Biobricks and making the miniprep, make the digestion mix.
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</p>
</p>
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<p class="title1">Checking of the genetic constructions  </p>
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<p class="title1 " id="select5">Checking of the genetic constructions  </p>
<p class="texte">
<p class="texte">
1) Colony PCR
1) Colony PCR
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<br/>The sequencing of the genetic constructions was performed by Eurofins Genomics Company by mixing 15µL of pure plasmid solution with 2µL of one primer.
<br/>The sequencing of the genetic constructions was performed by Eurofins Genomics Company by mixing 15µL of pure plasmid solution with 2µL of one primer.
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<p class="title1"> <I>B. subtilis</I> transformation</p>
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<p class="title1" id="select6"> <I>B. subtilis</I> transformation</p>
<p class="texte">
<p class="texte">
<br/>Strain: <I>Bacillus subtilis</I> 168.  
<br/>Strain: <I>Bacillus subtilis</I> 168.  
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<br>The complete mixture should be dissolved in 100 ml. First add 50 ml milliQ water and mix. When everything is dissolved add MQ water till 100 ml. Filter sterilize the complete mixture and store at -20°C.
<br>The complete mixture should be dissolved in 100 ml. First add 50 ml milliQ water and mix. When everything is dissolved add MQ water till 100 ml. Filter sterilize the complete mixture and store at -20°C.
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<p class="title1"> Checking of the genetic constructions after plasmid integration in <I>Bacillus subtilis</I> </p>
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<p class="title1" id="select7"> Checking of the genetic constructions after plasmid integration in <I>Bacillus subtilis</I> </p>
<p class="texte">
<p class="texte">
<br>Test of the pSBBS4S plasmid integration in Bacillus subtilis genome on the threonine site:
<br>Test of the pSBBS4S plasmid integration in Bacillus subtilis genome on the threonine site:
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Moreover, a colony PCR can be performed with the same protocol as previously presented. The used primers are VFBS and VRBS.
Moreover, a colony PCR can be performed with the same protocol as previously presented. The used primers are VFBS and VRBS.
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<p class="title1">Final Tests</p>
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<p class="title1" id="select8">Final Tests</p>
<p class="texte">
<p class="texte">
<B> Chemotaxis test </B>
<B> Chemotaxis test </B>
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<br>The next step begins with preparation of the fungal samples.PDA cores containing the fungal culture is crushed and left in culture in the PDB . Then it pass through a filter of 100 µm (to remove large aggregates) and  through a 40 µm filter to allow small molecules. The caught hyphae are placed in the PDB for 24 to 48 hours. This liquid culture is filtered to retain the living fragment and place in solution. Then, 2.5 of OD is adjusted. Obtained leaves above are detached from the plants using a scalpel and placed in boxes above wet absorbent paper (leaves are kept alive for a week). Above each leaf is deposited 5μL of fungal solution (using beveled tips because it is too viscous), as control we keep inoculated leaf without fungus. Pictures are achieved at different times. All the plants are destroyed by autoclaving.
<br>The next step begins with preparation of the fungal samples.PDA cores containing the fungal culture is crushed and left in culture in the PDB . Then it pass through a filter of 100 µm (to remove large aggregates) and  through a 40 µm filter to allow small molecules. The caught hyphae are placed in the PDB for 24 to 48 hours. This liquid culture is filtered to retain the living fragment and place in solution. Then, 2.5 of OD is adjusted. Obtained leaves above are detached from the plants using a scalpel and placed in boxes above wet absorbent paper (leaves are kept alive for a week). Above each leaf is deposited 5μL of fungal solution (using beveled tips because it is too viscous), as control we keep inoculated leaf without fungus. Pictures are achieved at different times. All the plants are destroyed by autoclaving.
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Revision as of 12:10, 15 October 2014