Team:ULB-Brussels/mat

From 2011.igem.org

(Difference between revisions)
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   <li><strong>PKD46-FOR: </strong></li>
   <li><strong>PKD46-FOR: </strong></li>
</ul>
</ul>
-
<p><strong>5’-  </strong>gagcaaaaggccagcaaaaggccaggaaccgtaaaaaggctgccacctgcatcgatttat-3’<strong></strong></p>
+
<p>5’-  <span class="vert">gagcaaaaggccagcaaaaggccaggaaccgtaaaaaggc</span>tgccacctgcatcgatttat-3’</p>
<ul>
<ul>
   <li><strong>PKD46-REV: </strong></li>
   <li><strong>PKD46-REV: </strong></li>
</ul>
</ul>
-
<p><strong>5’-</strong>cgtgagttttcgttccactgagcgtcagaccccgtagaaagagttttcgttccactgagc-3’<strong></strong></p>
+
<p><strong>5’-</strong><span class="vert">cgtgagttttcgttccactgagcgtcagaccccgtagaaa</span>gagttttcgttccactgagc-3’</p>
<ul>
<ul>
   <li><strong>PFL-FOR</strong>: </li>
   <li><strong>PFL-FOR</strong>: </li>
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   <li><strong>PCP-FOR:</strong></li>
   <li><strong>PCP-FOR:</strong></li>
</ul>
</ul>
-
<p>5’-tggctcttgtatctatcagtgaagcatcaagactaacaaatcagccaaacgtctcttcag-3’</p>
+
<p>5’-<span class="vert">tggctcttgtatctatcagtgaagcatcaagactaacaaa</span>tcagccaaacgtctcttcag-3’</p>
<ul>
<ul>
   <li><strong>PCP-REV: </strong></li>
   <li><strong>PCP-REV: </strong></li>
</ul>
</ul>
-
<p><strong>5’-</strong>ggggctgtatgcacaaagcatcttctgttgagttaagaacttatatgcgtctatttatgtagg-3’<strong></strong><br />
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<p><strong>5’-</strong><span class="vert">ggggctgtatgcacaaagcatcttctgttgagttaagaac</span>ttatatgcgtctatttatgtagg-3’</p>
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   In green is the 40 homologous nucleotides need for the yeast cloning.</p>
+
<p><strong><br />
 +
   In green is the 40 homologous nucleotides need for the yeast cloning.</strong></p>
<h2>Yeast cloning verification:</h2>
<h2>Yeast cloning verification:</h2>
<ul>
<ul>
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<h2>Biobrick frt’-cm-frt’</h2>
<h2>Biobrick frt’-cm-frt’</h2>
<ul>
<ul>
-
   <li><strong>frt’-cm-frt’-for</strong>: 5’-Gaagttcctatactttttagagaataggaacttcgttgatcgggcacgtaagagg-3’ </li>
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   <li><strong>frt’-cm-frt’-for</strong>:</li>
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  <li><strong>frt’-cm-frt’-rev&nbsp;</strong>:5’-GAAGTTCCTATTCTCTAAAAAGTATAGGAACTTCTTATTACGCCCCGCCCTGCC-3’</li>
+
</ul>
</ul>
-
<p>In green is the frt’ sequence which is not homologous with the chloramphenicol gene.</p>
+
<p> 5’-<span class="vert">gaagttcctatactttttagagaataggaacttc</span>gttgatcgggcacgtaagagg-3’ </p>
<ul>
<ul>
-
   <li><strong>topo-frt’-cm-frt’-for:</strong> tccggcaaaaaagggcaaggtgtcaccaccctgcccttttCgccagtgtgctggatttc</li>
+
   <li><strong>frt’-cm-frt’-rev&nbsp;</strong>:
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  <li><strong>topo-frt’-cm-frt’-rev:</strong> TGCAGCGGCCGCTACTAGTACTCTAGAAGCGGCCGCGAATGATGGATATCTGCAGATTTC </li>
+
    <p>5’-<span class="vert">gaagttcctattctctaaaaagtataggaacttc</span>ttattacgccccgccctgcc-3’</p>
 +
  </li>
</ul>
</ul>
-
<p>In red is the iGEM restriction sites (prefix and suffix) and in green is the mutation made to suppress the EcoRI restriction site (in the Topo® XL PCR plasmid).</p>
+
<p><strong>In green is the frt’ sequence which is not homologous with the chloramphenicol gene.</strong></p>
 +
<ul>
 +
  <li><strong>topo-frt’-cm-frt’-for:</strong> </li>
 +
</ul>
 +
<p><span class="black">5'-</span><span class="red">tccggcaaaaaagggcaaggtgtcaccaccctgccctttt</span>cgccagtgtgctgga<span class="vert">t</span>ttc-3'</p>
 +
<ul>
 +
  <li><strong>topo-frt’-cm-frt’-rev:</strong> </li>
 +
</ul>
 +
<p>5’-<span class="red">tgcagcggccgctactagtactctagaagcggccgcgaa</span>tgatggatatctgcaga<span class="vert">t</span>ttc-3’</p>
 +
<p><strong>In red is the iGEM restriction sites (prefix and suffix) and in green is the mutation made to suppress the EcoRI restriction site (in the Topo® XL PCR plasmid).</strong></p>
<h2>Biobrick frt’-cm-frt’ sequencing (primer M13 as described in Topo® XL PCR Kit)</h2>
<h2>Biobrick frt’-cm-frt’ sequencing (primer M13 as described in Topo® XL PCR Kit)</h2>
<ul>
<ul>
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</ul>
</ul>
<p> 5’-gaccggcagcaaatg-3’<strong></strong></p>
<p> 5’-gaccggcagcaaatg-3’<strong></strong></p>
-
 
+
</div>
-
 
+
</div>
</div>

Revision as of 13:16, 21 September 2011

Materials & Method: page1

Primers

The following primers were produced by Sigma-Aldrich (option desalt)

Yeast cloning:

  • PKD46-FOR:

5’-  gagcaaaaggccagcaaaaggccaggaaccgtaaaaaggctgccacctgcatcgatttat-3’

  • PKD46-REV:

5’-cgtgagttttcgttccactgagcgtcagaccccgtagaaagagttttcgttccactgagc-3’

  • PFL-FOR:

5’-Gcctttttacggttcctggc-3’

  • PFL-REV:

5’-tttctacgggggtctgacgc-3’

  • PCP-FOR:

5’-tggctcttgtatctatcagtgaagcatcaagactaacaaatcagccaaacgtctcttcag-3’

  • PCP-REV:

5’-ggggctgtatgcacaaagcatcttctgttgagttaagaacttatatgcgtctatttatgtagg-3’


In green is the 40 homologous nucleotides need for the yeast cloning.

Yeast cloning verification:

  • FLP/CI-FOR:

5’-Acatggcgagttttgacgag-3’

  • FLP/CI-REV:

5’-accacactagagaacatactg-3’

pINDEL sequencing:

  • pID-seq1:

5’-aggatcttcacctagatcctt-3’

  • pID-seq2:

 5’-gatgggctagtcaatgataatta-3’

  • pID-seq3:

5’-ccgttacgtaggtaggaatc-3’

  • pID-seq4:

5’-agatggggatggggcagtc-3’

  • pID-seq5:

5’-gatttcggatcaacgttcttaat-3’

  • pID-seq6:

5’-caatcactttcgtctactcc-3’

  • pID-seq7:

 5’-ccagatatttcgccgcgac-3’

  • pID-seq8:

5’-cggggccagcaaaaaatcca-3’

  • pID-seq9:

5’-ccctgatttttcaccacccc-3’

  • pID-seq10:

5’-cttccgaaaatgcaacgcga-3’

Biobrick frt’-cm-frt’

  • frt’-cm-frt’-for:

5’-gaagttcctatactttttagagaataggaacttcgttgatcgggcacgtaagagg-3’

  • frt’-cm-frt’-rev :

    5’-gaagttcctattctctaaaaagtataggaacttcttattacgccccgccctgcc-3’

In green is the frt’ sequence which is not homologous with the chloramphenicol gene.

  • topo-frt’-cm-frt’-for:

5'-tccggcaaaaaagggcaaggtgtcaccaccctgcccttttcgccagtgtgctggatttc-3'

  • topo-frt’-cm-frt’-rev:

5’-tgcagcggccgctactagtactctagaagcggccgcgaatgatggatatctgcagatttc-3’

In red is the iGEM restriction sites (prefix and suffix) and in green is the mutation made to suppress the EcoRI restriction site (in the Topo® XL PCR plasmid).

Biobrick frt’-cm-frt’ sequencing (primer M13 as described in Topo® XL PCR Kit)

  • FRT’-cm-FRT’-SEQ-FOR:

5’-Gaggaaacagctatga-3’

  • FRT’-cm-FRT’-SEQ-REV:

 5’-gaccggcagcaaatg-3’

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