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Team:ULB-Brussels/mat
From 2011.igem.org
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<h2>Biobrick frt’-cm-frt’</h2> | <h2>Biobrick frt’-cm-frt’</h2> | ||
<ul> | <ul> | ||
| - | <li><strong> | + | <li><strong>FRT’-CM-FRT’-FOR</strong>:</li> |
</ul> | </ul> | ||
<p> 5’-<span class="vert">gaagttcctatactttttagagaataggaacttc</span>gttgatcgggcacgtaagagg-3’ </p> | <p> 5’-<span class="vert">gaagttcctatactttttagagaataggaacttc</span>gttgatcgggcacgtaagagg-3’ </p> | ||
<ul> | <ul> | ||
| - | <li><strong> | + | <li><strong>FRT'-CM-FRT'-REV </strong>: |
<p>5’-<span class="vert">gaagttcctattctctaaaaagtataggaacttc</span>ttattacgccccgccctgcc-3’</p> | <p>5’-<span class="vert">gaagttcctattctctaaaaagtataggaacttc</span>ttattacgccccgccctgcc-3’</p> | ||
</li> | </li> | ||
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<p><strong>In green is the frt’ sequence which is not homologous with the chloramphenicol gene.</strong></p> | <p><strong>In green is the frt’ sequence which is not homologous with the chloramphenicol gene.</strong></p> | ||
<ul> | <ul> | ||
| - | <li><strong> | + | <li><strong>TOPO-FRT'-CM-FRT'-FOR:</strong> </li> |
</ul> | </ul> | ||
<p><span class="black">5'-</span><span class="red">tccggcaaaaaagggcaaggtgtcaccaccctgccctttt</span>cgccagtgtgctgga<span class="vert">t</span>ttc-3'</p> | <p><span class="black">5'-</span><span class="red">tccggcaaaaaagggcaaggtgtcaccaccctgccctttt</span>cgccagtgtgctgga<span class="vert">t</span>ttc-3'</p> | ||
<ul> | <ul> | ||
| - | <li><strong> | + | <li><strong>TOPO-FRT'-CM-FRT'-REV:</strong></li> |
</ul> | </ul> | ||
<p>5’-<span class="red">tgcagcggccgctactagtactctagaagcggccgcgaa</span>tgatggatatctgcaga<span class="vert">t</span>ttc-3’</p> | <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> | <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 | + | <h2>Biobrick FRT'-CM-FRT' sequencing (primer M13 as described in Topo® XL PCR Kit)</h2> |
<ul> | <ul> | ||
| - | <li><strong>FRT’- | + | <li><strong>FRT’-CM-FRT’-SEQ-FOR: </strong></li> |
</ul> | </ul> | ||
<p>5’-Gaggaaacagctatga-3’</p> | <p>5’-Gaggaaacagctatga-3’</p> | ||
<ul> | <ul> | ||
| - | <li><strong>FRT’- | + | <li><strong>FRT’-CM-FRT’-SEQ-REV:</strong></li> |
</ul> | </ul> | ||
<p> 5’-gaccggcagcaaatg-3’<strong></strong></p> | <p> 5’-gaccggcagcaaatg-3’<strong></strong></p> | ||
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Revision as of 13:25, 21 September 2011
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’
