Team:SDU-Denmark/primers

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= Our Parts =
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<groupparts>iGEM010 SDU-Denmark</groupparts>
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<br>
== '''Primers''' ==
== '''Primers''' ==
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<p style="text-align: justify;">
<br>
<br>
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On this page we list all the primers we designed and used during the summer. Numbers in paranthesis is basic temperature (celcius) <br><br>
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On this page we list all the primers we designed and used during the summer. All primers were kindly provided by [http://www.dna-technology.dk/ DNA Technology]. <br><br>
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'''''Primers for FlhDC master operon'''''
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In the following, coding sequences are colored <span
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style="color: rgb(88, 115, 248);">blue</span>, restriction sites are colored <span
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style="color: rgb(80, 204, 56);">green</span> and nucleotides that are both are shown in <span
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style="color: rgb(255, 153, 0);">orange</span>.<br>
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The mutation sites in the FlhDC mutation primers are shown in <span
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style="color: rgb(205, 51, 204);">purple</span>.
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<br><br>
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<html>
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<head>
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</head>
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<body>
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<table style="text-align: left; width: 630px; height: 508px;"
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border="1" cellpadding="2" cellspacing="2">
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  <tbody>
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    <tr>
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      <td
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style="font-family: Helvetica,Arial,sans-serif; height: 44px; width: 60px;">Primer
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name</td>
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      <td
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style="font-family: Helvetica,Arial,sans-serif; height: 44px; width: 638px;">Sequence</td>
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      <td
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style="width: 51px; font-family: Helvetica,Arial,sans-serif; height: 44px;">Tm
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(C)</td>
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    </tr>
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    <tr>
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      <td
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style="font-family: Helvetica,Arial,sans-serif; height: 44px; width: 60px;">FlhDC
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fw</td>
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      <td
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style="font-family: Courier New; height: 44px; width: 638px;">5'-GTTTCTTC<span
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style="color: rgb(80, 204, 56);">GAATTC</span>GCGGCCGCT<span
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style="color: rgb(80, 204, 56);">TCTAG</span><span
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style="color: rgb(88, 115, 248);"><span
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style="color: rgb(255, 153, 0);">A</span>TGCATACCTCCGAGTTGCTGAAA</span>-3'</td>
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      <td
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style="width: 51px; font-family: Helvetica,Arial,sans-serif; height: 44px;">88.3</td>
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    </tr>
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    <tr>
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      <td
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style="font-family: Helvetica,Arial,sans-serif; height: 44px; width: 60px;">FlhDC
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rv</td>
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      <td
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style="font-family: Courier New; height: 44px; width: 638px;">5'-GTTTCTTC<span
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style="color: rgb(80, 204, 56);">CTGCAG</span>CGGCCGCTACTATT<span
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style="color: rgb(88, 115, 248);">AAACAGCCTGTACTCTCTGTTC</span>-3'</td>
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      <td
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style="width: 51px; font-family: Helvetica,Arial,sans-serif; height: 44px;">88.3</td>
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    </tr>
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    <tr>
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      <td
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style="font-family: Helvetica,Arial,sans-serif; height: 44px; width: 60px;">FlhDC
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mutation fw*</td>
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      <td
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style="font-family: Courier New; height: 44px; width: 638px;">5'-<span
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style="color: rgb(88, 115, 248);">TTGGCAGCTTTGCC<span
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style="color: rgb(204, 51, 204);">C</span>GCAGCTTATG</span>-3'</td>
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      <td
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style="width: 51px; font-family: Helvetica,Arial,sans-serif; height: 44px;">61.0</td>
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    </tr>
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    <tr>
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      <td
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style="font-family: Helvetica,Arial,sans-serif; height: 44px; width: 60px;">FlhDC
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mutation rv*</td>
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      <td
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style="font-family: Courier New; height: 44px; width: 638px;">5'-<span
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style="color: rgb(88, 115, 248);">CATAAGCTGC<span
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style="color: rgb(204, 51, 204);">G</span>GGCAAAGCTGCCAA</span>-3'</td>
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      <td
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style="width: 51px; font-family: Helvetica,Arial,sans-serif; height: 44px;">61.0</td>
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    </tr>
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    <tr>
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      <td
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style="font-family: Helvetica,Arial,sans-serif; height: 44px; width: 60px;">Photosensor
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fw</td>
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      <td
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style="font-family: Courier New; height: 44px; width: 638px;">5'-GTTTCTTC<span
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style="color: rgb(80, 204, 56);">GAATTC</span>GCGGCCGCT<span
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style="color: rgb(80, 204, 56);">TCTAG</span><span
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style="color: rgb(88, 115, 248);"><span
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style="color: rgb(255, 102, 0);">A</span>TGGTGGGACTTACGACCTC</span>-3'</td>
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      <td
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style="width: 51px; font-family: Helvetica,Arial,sans-serif; height: 44px;">73.1</td>
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    </tr>
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    <tr>
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      <td
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style="font-family: Helvetica,Arial,sans-serif; height: 44px; width: 60px;">Photosensor
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rv</td>
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      <td
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style="font-family: Courier New; height: 44px; width: 638px;">5'-GTTTCTTC<span
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style="color: rgb(80, 204, 56);">CTGCAG</span>CGGCCGCT<span
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style="color: rgb(80, 204, 56);">ACTAGT</span>A<span
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style="color: rgb(88, 115, 248);">TCAGAAGGTTTCCCAGTTCGCATC</span>-3'</td>
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      <td
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style="width: 51px; font-family: Helvetica,Arial,sans-serif; height: 44px;">73.9</td>
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    </tr>
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    <tr>
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      <td
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style="font-family: Helvetica,Arial,sans-serif; height: 44px; width: 60px;">NinaB
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fw</td>
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      <td
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style="font-family: Courier New; height: 44px; width: 638px;">5'-<span
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style="color: rgb(80, 204, 56);"><span
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style="color: black;">GTTTCTTC</span>GAATTC</span>GCGGCCGCT<span
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style="color: rgb(80, 204, 56);">TCTAG</span><span
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style="color: rgb(88, 115, 248);"><span
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style="color: rgb(255, 102, 0);">A</span>TGGCAGCCGGTGTCTTCAAGAG</span>-3'</td>
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      <td
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style="width: 51px; font-family: Helvetica,Arial,sans-serif; height: 44px;">73.1</td>
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    </tr>
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    <tr>
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      <td
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style="font-family: Helvetica,Arial,sans-serif; height: 44px; width: 60px;">NinaB
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rv</td>
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      <td
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style="font-family: Courier New; height: 44px; width: 638px;">5'-<span
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style="color: rgb(80, 204, 56);"><span
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style="color: black;">GTGTCTTC</span>CTGCAG</span>CGGCCGCT<span
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style="color: rgb(80, 204, 56);">ACTAGT</span>A<span
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style="color: rgb(88, 115, 248);">CTAAATGGCATTGGGTGCAAACCATC</span>-3'</td>
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      <td
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style="width: 51px; font-family: Helvetica,Arial,sans-serif; height: 44px;">72.4</td>
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    </tr>
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    <tr>
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      <td
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style="font-family: Helvetica,Arial,sans-serif; height: 44px; width: 60px;">NinaB
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2 fw**</td>
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      <td
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style="font-family: Courier New; height: 44px; width: 638px;">5'-CCGCT<span
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style="color: rgb(80, 204, 56);">TCTAG</span><span
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style="color: rgb(88, 115, 248);"><span
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style="color: rgb(255, 102, 0);">A</span>TGGCAGCCGGTGTCTTCAAGAG</span>-3'</td>
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      <td
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style="width: 51px; font-family: Helvetica,Arial,sans-serif; height: 44px;">68.1</td>
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    </tr>
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    <tr>
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      <td
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style="font-family: Helvetica,Arial,sans-serif; height: 44px; width: 60px;">NinaB
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2 rv**</td>
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      <td
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style="font-family: Courier New; height: 44px; width: 638px;">5'-CGCT<span
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style="color: rgb(80, 204, 56);">ACTAGT</span>A<span
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style="color: rgb(88, 115, 248);">CTAAATGGCATTGGGTGCAAACCATC</span>-3'</td>
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      <td
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style="width: 51px; font-family: Helvetica,Arial,sans-serif; height: 44px;">65.6</td>
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    </tr>
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  </tbody>
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</table>
<br>
<br>
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We needed to design 4 primers for this part. The FlhDC operon contains a PST1 restriction site, therefore we wanted to introduce a silent mutation, C --> T, at position 822 in the operon.<br><br>
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</body>
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''Reverse primer w. mutation at position 13 (gene position 822):'' 5’-CATAAGCTGCGGTCAAAGCTGCCAA -3’ (59)<br><br>
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</html>
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''Forward primer w. mutation at position 13 (gene position 822):'' 5’-TTGGCAGCTTTGACCGCAGCTTATG-3’        (59)<br><br>
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Lastly we added primers with prefix and suffix to the PCR result of the above:<br><br>
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[[FlhDC forward:]]<br>
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(*) Mutation primers were designed to introduce a silent mutation at position 822 in the operon to eliminate an illegal PstI site.<br>
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''Prefixprimer:'' 5'-G TTT CTT CGA ATT CGC GGC CGC TTC TAG ATG CAT ACC TCC GAG TTG CTG AAA-3' (Coding sequence (CS): 56) (Primer total (PT): 72)<br><br>
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(**) Due to the formation of hairpin structures of primers ninaB fw and ninaB rv, new primers were designed containing only the innermost restriction sites (XbaI and SpeI).<br><br>
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[[FlhDC reverse:]]<br>
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''Suffixprimer:'' 5'-GT TTC TTC CTG CAG CGG CCG CTA CTA TTA AAC AGC CTG TAC TCT CTG TTC-3' (CS: 56) (PT: 72)<br><br>
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</p>
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'''''SopIItrs'''''
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<br>
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''Forward primer:''  5’-GTTTCTTCGAATTCGCGGCCGCTTCTAGATGGTGGGACTTACGACC-3’ (CS: 50) (PT: 73)<br><br>
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''Reverse primer:''  5’-GTTTCTTCCTGCAGCGGCCGCTACTAGTATTAAAATGTTTCCCAGTTCT-3’ (CS: 70) (PT: 44) <br><br>
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Latest revision as of 21:47, 27 October 2010

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