Difference between revisions of "Team:Valencia UPV/Notebook"

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<div class="container">
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<div class="container">
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<div class="timeline">
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<div class="timeline">
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<div class="timeline-hline"></div>
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<div class="blog-post-item">
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<div class="timeline-entry rounded">
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      18<span>May</span>
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18<span>May</span>
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      <div class="timeline-vline"></div>
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<div class="timeline-vline"></div>
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  </div>
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</div>
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  <ul class="blog-post-info list-inline">
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<ul class="blog-post-info list-inline">
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      <li><span class="font-lato">May 18, 2016</span></li>
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<li><span class="font-lato">May 18, 2016</span></li>
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  </ul>
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</ul>
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  <p><a href=" glycerinated cultures of C58 <i>Agrobacterium</i> with dsRED from GoldenBraid Collection. Prepare broth culture (5 mL LB + 5 &mu"></a>L kanamycin + 5 μL Rifampin) 1:1000 and incubate overnight at 28^C. <br></p>
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<p>Take glycerinated cultures of C58 <i>Agrobacterium</i> with dsRED from
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</div>
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GoldenBraid Collection. Prepare broth culture (5 mL LB + 5 μL kanamycin +
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<div class="blog-post-item">
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5 μL Rifampin) 1:1000 and incubate overnight at 28^C.<br></p>
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  <div class="timeline-entry rounded">
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</div>
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      19<span>May</span>
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<div class="blog-post-item">
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      <div class="timeline-vline"></div>
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<div class="timeline-entry rounded">
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  </div>
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19<span>May</span>
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  <ul class="blog-post-info list-inline">
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<div class="timeline-vline"></div>
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      <li><span class="font-lato">May 19, 2016</span></li>
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</div>
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  </ul>
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<ul class="blog-post-info list-inline">
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  <p><a href="esh previously made culture by inoculating 10 &mu"></a>L in a new culture medium.<br></p>
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<li><span class="font-lato">May 19, 2016</span></li>
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</div>
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</ul>
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<div class="blog-post-item">
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<p>Refresh previously made culture by inoculating 10 μL in a new culture
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  <div class="timeline-entry rounded">
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medium.<br></p>
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      20<span>May</span>
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</div>
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      <div class="timeline-vline"></div>
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<div class="blog-post-item">
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  </div>
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<div class="timeline-entry rounded">
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  <ul class="blog-post-info list-inline">
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20<span>May</span>
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      <li><span class="font-lato">May 20, 2016</span></li>
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<div class="timeline-vline"></div>
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  </ul>
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</div>
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  <p><a href="#">Agroinfiltration</a> in <i>Nicotiana benthamiana</i> of C58 with dsRED.<br></p>
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<ul class="blog-post-info list-inline">
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</div>
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<li><span class="font-lato">May 20, 2016</span></li>
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<div class="blog-post-item">
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</ul>
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  <div class="timeline-entry rounded">
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<p><a href="#">Agroinfiltration</a> in <i>Nicotiana benthamiana</i> of C58
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      06<span>Jun</span>
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with dsRED.<br></p>
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      <div class="timeline-vline"></div>
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</div>
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  </div>
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<div class="blog-post-item">
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  <ul class="blog-post-info list-inline">
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<div class="timeline-entry rounded">
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      <li><span class="font-lato">June 06, 2016</span></li>
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06<span>Jun</span>
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  </ul>
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<div class="timeline-vline"></div>
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  <p></p>
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</div>
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  <ul>
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<ul class="blog-post-info list-inline">
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      <li>
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<li><span class="font-lato">June 06, 2016</span></li>
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        href="
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</ul>
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        <l>
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<p></p>
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        T"></a>
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<ul>
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        <l>
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<li>Take from the glycerinates of Goldenbraid Collection:</li>
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        Take from the glycerinates of Goldenbraid Collection:
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</ul>
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      </li>
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<p><br></p>
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  </ul>
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<div class="table-responsive" style="width:55%;overflow:inherit">
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  <p><a href=""></a><br></p>
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<table class="table table-bordered table-striped">
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  <div class="table-responsive" style="width:55%;overflow:inherit">
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<tr>
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      <table class="table table-bordered table-striped">
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<td>Plasmid</td>
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        <tr>
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<td>GB Code</td>
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            <td><a href="Plas"></a>Plasmid</td>
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</tr>
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            <td>GB Code</td>
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<tr>
−
        </tr>
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<td>pD6B3 α1</td>
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        <tr>
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<td>GB0015</td>
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            <td><a href="3 &alpha"></a>1</td>
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</tr>
−
            <td>GB0015</td>
+
<tr>
−
        </tr>
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<td>pD6B3 α2</td>
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        <tr>
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<td>GB0017</td>
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            <td><a href="3 &alpha"></a>2</td>
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</tr>
−
            <td>GB0017</td>
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<tr>
−
        </tr>
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<td>pD6B3 Ω1</td>
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        <tr>
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<td>GB0019</td>
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            <td><a href="3 &Omega"></a>1</td>
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</tr>
−
            <td>GB0019</td>
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<tr>
−
        </tr>
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<td>pD6B3 Ω2</td>
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        <tr>
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<td>GB0021</td>
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            <td><a href="3 &Omega"></a>2</td>
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</tr>
−
            <td>GB0021</td>
+
<tr>
−
        </tr>
+
<td>pUPD2</td>
−
        <tr>
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<td>GB0307</td>
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            <td><a href="pUPD"></a>pUPD2</td>
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</tr>
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            <td>GB0307</td>
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</table>
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        </tr>
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</div>
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      </table>
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<p><br>
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  </div>
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Prepare liquid culture (3 mL LB + 3 μL antibiotic) 1:1000. Incubate at
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  <p><a href=""></a><br><a href="are liquid culture (3 mL LB + 3 &mu"></a>L antibiotic) 1:1000. Incubate at 37^C overnight. <br><a href=""></a><br></p>
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37^C overnight.<br>
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  <ul>
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<br></p>
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      <li>
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<ul>
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        href="
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<li>Experiment with snails:</li>
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        <l>
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</ul>
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        E"></a>
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<p>Two experiments: one with infiltrated <i>Nicotiana benthamiana</i> and
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        <l>
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another with not infiltrated N.benthamiana (negative control). Lettuce
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        Experiment with snails:
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leafs haven’t been correctly infiltrated and it seems that the expression
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      </li>
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level is low.<br>
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  </ul>
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Let both <i>N. benthamiana</i> leafs with snails overnight at room
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  <p><a href="experiments: one with infiltrated <i>Nicotiana benthamiana</i> and another with not infiltrated N.benthamiana (negative control). Lettuce leafs haven&rsquo"></a>t been correctly infiltrated and it seems that the expression level is low.<br><a href="Let "></a>Let both <i>N. benthamiana</i> leafs with snails overnight at room temperature in separated boxes. We will observe if snails eat the leafs and if appears fluorescence. <br><a href=""></a><br></p>
+
temperature in separated boxes. We will observe if snails eat the leafs
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</div>
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and if appears fluorescence.<br>
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<div class="blog-post-item">
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<br></p>
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  <div class="timeline-entry rounded">
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</div>
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      15<span>Jun</span>
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<div class="blog-post-item">
−
      <div class="timeline-vline"></div>
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<div class="timeline-entry rounded">
−
  </div>
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15<span>Jun</span>
−
  <ul class="blog-post-info list-inline">
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<div class="timeline-vline"></div>
−
      <li><span class="font-lato">June 15, 2016</span></li>
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</div>
−
  </ul>
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<ul class="blog-post-info list-inline">
−
  <p><a href="riment is over due to snails haven&rsquo"></a>t eaten leafs enough so we have not been able to see fluorescence.<br></p>
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<li><span class="font-lato">June 15, 2016</span></li>
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</div>
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</ul>
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<div class="blog-post-item">
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<p>Experiment is over due to snails haven’t eaten leafs enough so we have
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  <div class="timeline-entry rounded">
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not been able to see fluorescence.<br></p>
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      30<span>Jun</span>
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</div>
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      <div class="timeline-vline"></div>
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<div class="blog-post-item">
−
  </div>
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<div class="timeline-entry rounded">
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  <ul class="blog-post-info list-inline">
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30<span>Jun</span>
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      <li><span class="font-lato">June 30, 2016</span></li>
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<div class="timeline-vline"></div>
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  </ul>
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</div>
−
  <p><a href="Oran"></a>Orange DNA Genome Extraction protocol href<br><a href=""></a><br></p>
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<ul class="blog-post-info list-inline">
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  <ul>
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<li><span class="font-lato">June 30, 2016</span></li>
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      <li>
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</ul>
−
        href="
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<p>Orange DNA Genome Extraction protocol href<br>
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        <l>
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<br></p>
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        T"></a>
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<ul>
−
        <l>
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<li>Take from the glycerinates of GoldenBraid Collection:</li>
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        Take from the glycerinates of GoldenBraid Collection:
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</ul>
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      </li>
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<p><br></p>
−
  </ul>
+
<div class="table-responsive" style="width:55%;overflow:inherit">
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  <p><a href=""></a><br></p>
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<table class="table table-bordered table-striped">
−
  <div class="table-responsive" style="width:55%;overflow:inherit">
+
<tr>
−
      <table class="table table-bordered table-striped">
+
<td>Plasmid</td>
−
        <tr>
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<td>GB Code</td>
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            <td><a href="Plas"></a>Plasmid</td>
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</tr>
−
            <td>GB Code</td>
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<tr>
−
        </tr>
+
<th>omoter 35s:Cas9:nopaline synthase terminator (Tnos)</th>
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        <tr>
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<th>GB0639</th>
−
            <th> href="Prom"></a>Promoter 35s:Cas9:nopaline synthase terminator (Tnos)</th>
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</tr>
−
            <th>GB0639</th>
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<tr>
−
        </tr>
+
<td>Luciferase (Luc) in pUPD2</td>
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        <tr>
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<td>GB0096</td>
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            <td><a href="Luci"></a>Luciferase (Luc) in pUPD2</td>
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</tr>
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            <td>GB0096</td>
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<tr>
−
        </tr>
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<td>Tnos in pUPD2</td>
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        <tr>
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<td>GB0037</td>
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            <td><a href="Tnos"></a>Tnos in pUPD2</td>
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</tr>
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            <td>GB0037</td>
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</table>
−
        </tr>
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</div>
−
      </table>
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<p><br></p>
−
  </div>
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</div>
−
  <p><a href=""></a><br></p>
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<div class="blog-post-item">
−
</div>
+
<div class="timeline-entry rounded">
−
<div class="blog-post-item">
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01<span>Jul</span>
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  <div class="timeline-entry rounded">
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<div class="timeline-vline"></div>
−
      01<span>Jul</span>
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</div>
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      <div class="timeline-vline"></div>
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<ul class="blog-post-info list-inline">
−
  </div>
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<li><span class="font-lato">July 01, 2016</span></li>
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  <ul class="blog-post-info list-inline">
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</ul>
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      <li><span class="font-lato">July 01, 2016</span></li>
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<p></p>
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  </ul>
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<ul>
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  <p><a href="iniprep with E.Z.N.A ®"></a>.  Plasmid Mini Kit I, Q(capless) Spin of: <br></p>
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<li>Miniprep with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of:</li>
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  <ul>
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<li style="list-style: none; display: inline">
−
      <li>
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<ul>
−
        href="
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<li>&gt;Promoter 35s:Cas9 : Tnos</li>
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        <l>
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</ul>
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        "></a>
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<p></p>
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        <l>
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</li>
−
        Promoter 35s:Cas9 : Tnos
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<li></li>
−
      </li>
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<li style="list-style: none; display: inline">
−
  </ul>
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<ul>
−
  <p><a href=""></a><br><a href="uciferase and nopaline synthase terminator cultures haven&rsquo"></a>t succeed. Repeat Luc and Tnos cultures. <br>
+
<li>Luciferase and nopaline synthase terminator cultures haven’t
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  </ul>
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succeed. Repeat Luc and Tnos cultures.</li>
−
  <p><a href=""></a><br><a href="Prim"></a>Primers IG16JUN01 and IG16JUN02 have arrived.<br><a href=""></a><br><a href="Fini"></a>Finish orange DNA Genome Extraction and check DNA concentration with NanoDrop. Concentration is very low so extraction will be done again.<br><a href=""></a><br></p>
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</ul>
−
</div>
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<p></p>
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<div class="blog-post-item">
+
</li>
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  <div class="timeline-entry rounded">
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<li></li>
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      02<span>Jul</span>
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<li>Primers IG16JUN01 and IG16JUN02 have arrived.</li>
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      <div class="timeline-vline"></div>
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<li></li>
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  </div>
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<li>Finish orange DNA Genome Extraction and check DNA concentration with
−
  <ul class="blog-post-info list-inline">
+
NanoDrop. Concentration is very low so extraction will be done
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      <li><span class="font-lato">July 02, 2016</span></li>
+
again.</li>
−
  </ul>
+
</ul>
−
  <p><a href="iniprep with E.Z.N.A ®"></a>. Plasmid Mini Kit I, Q(capless) Spin of:<br></p>
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</div>
−
  <ul>
+
</div>
−
  <li>
+
</div>
−
      href="
+
</section>
−
      <l>
+
−
      "></a>
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−
      <l>
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−
      Luc in pUPD2
+
−
  </li>
+
−
  </p>
+
−
  <ul>
+
−
      <li>
+
−
        href="
+
−
        <l>
+
−
        "></a>
+
−
        <l>
+
−
        Tnos in pUPD2
+
−
      </li>
+
−
  </ul>
+
−
  <p><a href=""></a><br></p>
+
−
  <ul>
+
−
      <li>
+
−
        href="
+
−
        <l>
+
−
        C"></a>
+
−
        <l>
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−
        Check orange DNA genome concentration with NanoDrop.
+
−
      </li>
+
−
  </ul>
+
−
  <p><a href=""></a><br></p>
+
−
  <div class="table-responsive" style="width:55%;overflow:inherit">
+
−
      <table class="table table-bordered table-striped">
+
−
        <tr>
+
−
            <td><a href="le</td><td>DNA concentration (ng / &mu"></a>L)</td>
+
−
        </tr>
+
−
        <tr>
+
−
            <td><a href="Clem"></a>Clemenules1 </td>
+
−
            <td>3153.8</td>
+
−
        </tr>
+
−
        <tr>
+
−
            <td><a href="Clem"></a>Clemenules 2 </td>
+
−
            <td>4527.9</td>
+
−
        </tr>
+
−
      </table>
+
−
  </div>
+
−
  <p><a href=""></a><br></p>
+
−
  <ul>
+
−
      <li>
+
−
        href="
+
−
        <l>
+
−
        P"></a>
+
−
        <l>
+
−
        Perform a PCR to bind linker with luciferase:
+
−
      </li>
+
−
  </ul>
+
−
  <p></p>
+
−
  <div class="table-responsive" style="width:55%;overflow:inherit">
+
−
      <table class="table table-bordered table-striped">
+
−
        <tr>
+
−
            <td><a href="ent</td><td>Volume(&mu"></a>L)</td>
+
−
            <td>Program</td>
+
−
        </tr>
+
−
        <tr>
+
−
            <td><a href="Luci"></a>LuciferasepUPD</td>
+
−
            <td>1</td>
+
−
            <td>Temperature</td>
+
−
            <td>Time </td>
+
−
        </tr>
+
−
        <tr>
+
−
            <td><a href="Buff"></a>Buffer HF</td>
+
−
            <td>10</td>
+
−
            <td>98^C</td>
+
−
            <td>5 minutes</td>
+
−
        </tr>
+
−
        <tr>
+
−
            <td><a href="dNTP"></a>dNTPs</td>
+
−
            <td>2</td>
+
−
            <td>98^C</td>
+
−
            <td>35x</td>
+
−
            <td>30 seconds</td>
+
−
        </tr>
+
−
        <tr>
+
−
            <td><a href="IG16"></a>IG16JUN01</td>
+
−
            <td>2.5</td>
+
−
            <td>70^C</td>
+
−
            <td>35x</td>
+
−
            <td>30 seconds</td>
+
−
        </tr>
+
−
        <tr>
+
−
            <td><a href="IG16"></a>IG16JUN02</td>
+
−
            <td>2.5</td>
+
−
            <td>72^C</td>
+
−
            <td>35x</td>
+
−
            <td>1 minute 30 seconds</td>
+
−
        </tr>
+
−
        <tr>
+
−
            <td><a href="Taq "></a>Taq phusion</td>
+
−
            <td>0.5</td>
+
−
            <td>72^C</td>
+
−
            <td>10 minutes</td>
+
−
        </tr>
+
−
        <tr>
+
−
            <td><a href="b>2</sub>O milli-Q</td><td>31.5</td><td>16^C</td><td>&infin"></a></td>
+
−
        </tr>
+
−
      </table>
+
−
  </div>
+
−
  <p><a href=""></a><br></p>
+
−
</div>
+
−
<div class="blog-post-item">
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−
<div class="timeline-entry rounded">
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−
  04<span>Jul</span>
+
−
  <div class="timeline-vline"></div>
+
−
</div>
+
−
<ul class="blog-post-info list-inline">
+
−
  <li><span class="font-lato">July 04, 2016</span></li>
+
−
</ul>
+
−
<p><a href=""></a><br><a href="Run "></a>Run electrophoresis gel of Clemenules DNA (agarose 1%). 45 mL agarose gel at 1% 0.45 g of agarose with 45 mL of TAE 1X. Voltage used is 100 V.<br><a href=""></a><br><a href="Rice"></a>Rice DNA Genome Extraction Protocol href<br><a href=""></a><br><a href="Run "></a>Run electrophoresis gel of Luciferase PCR product. 45 mL agarose gel at 1% 0.45 g of agarose with 45 mL of TAE 1X. Voltage used is 100 V.<br><a href=""></a><br><a href="Liga"></a>Ligate Reaction --> Linker:Luciferase into a pUPD2 <br><a href=""></a>
+
  
 
</body>
 
</body>

Revision as of 11:57, 22 September 2016

18May
  • May 18, 2016

Take glycerinated cultures of C58 Agrobacterium with dsRED from GoldenBraid Collection. Prepare broth culture (5 mL LB + 5 μL kanamycin + 5 μL Rifampin) 1:1000 and incubate overnight at 28^C.

19May
  • May 19, 2016

Refresh previously made culture by inoculating 10 μL in a new culture medium.

20May
  • May 20, 2016

Agroinfiltration in Nicotiana benthamiana of C58 with dsRED.

06Jun
  • June 06, 2016

  • Take from the glycerinates of Goldenbraid Collection:


Plasmid GB Code
pD6B3 α1 GB0015
pD6B3 α2 GB0017
pD6B3 Ω1 GB0019
pD6B3 Ω2 GB0021
pUPD2 GB0307


Prepare liquid culture (3 mL LB + 3 μL antibiotic) 1:1000. Incubate at 37^C overnight.

  • Experiment with snails:

Two experiments: one with infiltrated Nicotiana benthamiana and another with not infiltrated N.benthamiana (negative control). Lettuce leafs haven’t been correctly infiltrated and it seems that the expression level is low.
Let both N. benthamiana leafs with snails overnight at room temperature in separated boxes. We will observe if snails eat the leafs and if appears fluorescence.

15Jun
  • June 15, 2016

Experiment is over due to snails haven’t eaten leafs enough so we have not been able to see fluorescence.

30Jun
  • June 30, 2016

Orange DNA Genome Extraction protocol href

  • Take from the glycerinates of GoldenBraid Collection:


Plasmid GB Code
omoter 35s:Cas9:nopaline synthase terminator (Tnos) GB0639
Luciferase (Luc) in pUPD2 GB0096
Tnos in pUPD2 GB0037


01Jul
  • July 01, 2016

  • Miniprep with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of:
    • >Promoter 35s:Cas9 : Tnos

    • Luciferase and nopaline synthase terminator cultures haven’t succeed. Repeat Luc and Tnos cultures.

  • Primers IG16JUN01 and IG16JUN02 have arrived.
  • Finish orange DNA Genome Extraction and check DNA concentration with NanoDrop. Concentration is very low so extraction will be done again.

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