Team:UNIPV-Pavia/Calendar/July/settimana3

From 2011.igem.org

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Digestions of previously purified plasmids were performed for ligations:
 
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</p>
 
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<p>CAMBIARE OGGETTI TABELLA</p>
 
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<center>
 
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<table border="1">
 
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    <tr>
 
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      <td><b>Plasmid</b></td>
 
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      <td><b>Kind</b></td>
 
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      <td><b>DNA (&mu;l)</b></td>
 
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      <td><b>H<small><sub>2</sub></small>O (&mu;l)</b></td>
 
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      <td><b>Enzyme 1 (&mu;l)</b></td>
 
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      <td><b>Enzyme 2 (&mu;l)</b></td>
 
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      <td><b>Buffer H (&mu;l)</b></td>
 
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      <td><b>Final Volume (&mu;l)</b></td>
 
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  </tr>
 
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  <tr>
 
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      <td></html><partinfo>BBa_C0060</partinfo><html></td>
 
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      <td>Insert</td>
 
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      <td>16.5</td>
 
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      <td>4</td>
 
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      <td>1 EcoRI</td>
 
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      <td>1 SpeI</td>
 
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      <td>2.5</td>
 
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      <td>25</td>
 
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  </tr>
 
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  <tr>
 
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      <td></html><partinfo>BBa_C0061</partinfo><html></td>
 
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      <td>Insert</td>
 
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      <td>13.2</td>
 
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      <td>7.3</td>
 
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      <td>1 XbaI</td>
 
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      <td>1 PstI</td>
 
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      <td>2.5</td>
 
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      <td>25</td>
 
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  </tr>
 
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  <tr>
 
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      <td></html><partinfo>BBa_K081022</partinfo><html></td>
 
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      <td>Insert</td>
 
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      <td>15.7</td>
 
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      <td>4.8</td>
 
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      <td>1 EcoRI</td>
 
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      <td>1 PstI</td>
 
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      <td>2.5</td>
 
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      <td>25</td>
 
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  </tr>
 
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  <tr>
 
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      <td></html><partinfo>BBa_B0030</partinfo><html></td>
 
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      <td>Vector</td>
 
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      <td>13.2</td>
 
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      <td>7.3</td>
 
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      <td>1 SpeI</td>
 
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      <td>1 PstI</td>
 
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      <td>2.5</td>
 
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      <td>25</td>
 
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  </tr>
 
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  <tr>
 
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      <td></html><partinfo>BBa_B0031</partinfo><html></td>
 
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      <td>Vector</td>
 
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      <td>12.4</td>
 
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      <td>8.1</td>
 
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      <td>1 SpeI</td>
 
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      <td>1 PstI</td>
 
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      <td>2.5</td>
 
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      <td>25</td>
 
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  </tr>
 
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  <tr>
 
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      <td></html><partinfo>BBa_B0032</partinfo><html></td>
 
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      <td>Vector</td>
 
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      <td>9.5</td>
 
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      <td>11</td>
 
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      <td>1 SpeI</td>
 
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      <td>1 PstI</td>
 
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      <td>2.5</td>
 
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      <td>25</td>
 
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  </tr>
 
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  <tr>
 
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      <td></html><partinfo>BBa_B0015</partinfo><html></td>
 
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      <td>Vector</td>
 
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      <td>7.9</td>
 
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      <td>12.6</td>
 
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      <td>1 EcoRI</td>
 
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      <td>1 XbaI</td>
 
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      <td>2.5</td>
 
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      <td>25</td>
 
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  </tr>
 
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  <tr>
 
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      <td></html><partinfo>pSB4C5</partinfo><html></td>
 
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      <td>Vector</td>
 
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      <td>3</td>
 
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      <td>17.5</td>
 
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      <td>1 EcoRI</td>
 
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      <td>1 PstI</td>
 
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      <td>2.5</td>
 
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      <td>25</td>
 
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  </tr>
 
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  <tr>
 
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      <td></html><partinfo>BBa_I13501</partinfo><html></td>
 
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      <td>Insert</td>
 
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      <td>7.2</td>
 
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      <td>13.3</td>
 
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      <td>1 XbaI</td>
 
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      <td>1 PstI</td>
 
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      <td>2.5</td>
 
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      <td>25</td>
 
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  </tr>
 
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</table>
 
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</center>
 
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<p>
 
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Reactions were incubated at 37°C for three hours while two medium-size agarose gels were prepared according to protocols.
 
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</p>
 
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<p>
 
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In the afternoon gel electrophoresis was performed.
 
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</p>
 
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</html>
 
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<html>
 
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<p>2 righe sopra inserire l' immagine</p>
 
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<p>
 
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As shown, in figure all clones were positive (apart from </html><partinfo>BBa_I13501</partinfo><html> run where we inexplicably didn't see any band), so we cut and purified the bands of interest. Cells harbouring </html><partinfo>BBa_I13501</partinfo><html> were again inoculated in 5 ml LB + Amp.
 
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</p>
 
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<p>
 
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After gel extraction, cut DNA was quantified:
 
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</p>
 
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<center>
 
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<table border="1">
 
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    <tr>
 
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      <td><b>Plasmid</b></td>
 
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      <td><b>DNA (ng/&mu;l)</b></td>
 
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  </tr>
 
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  <tr>
 
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      <td></html><partinfo>BBa_C0060</partinfo><html> (E-S)</td>
 
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      <td>3.9</td>
 
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  </tr>
 
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  <tr>
 
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      <td></html><partinfo>BBa_C0061</partinfo><html> (X-P)</td>
 
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      <td>4.1</td>
 
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  </tr>
 
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  <tr>
 
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      <td></html><partinfo>BBa_K081022</partinfo><html> (E-P)</td>
 
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      <td>4.4</td>
 
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  </tr>
 
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  <tr>
 
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      <td></html><partinfo>BBa_B0030</partinfo><html> (S-P)</td>
 
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      <td>10.3</td>
 
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  </tr>
 
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  <tr>
 
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      <td></html><partinfo>BBa_B0031</partinfo><html> (S-P)</td>
 
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      <td>11.8</td>
 
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  </tr>
 
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  <tr>
 
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      <td></html><partinfo>BBa_B0032</partinfo><html> (S-P)</td>
 
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      <td>10.1</td>
 
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  </tr>
 
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  <tr>
 
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      <td></html><partinfo>BBa_B0015</partinfo><html> (E-X)</td>
 
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      <td>12</td>
 
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  </tr>
 
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  <tr>
 
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      <td></html><partinfo>pSB4C5</partinfo><html> (E-P)</td>
 
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      <td>10.7</td>
 
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  </tr>
 
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</table>
 
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</center>
 
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<p>
 
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Then ligations were performed in a final volume of 10 &mu;l:
 
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</p>
 
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<center>
 
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<table border="1">
 
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    <tr>
 
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      <td><b>Ligation Name</b></td>
 
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      <td><b>Vector</b></td>
 
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      <td><b>Vector volume (&mu;l)</b></td>
 
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      <td><b>Insert</b></td>
 
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      <td><b>Insert volume (&mu;l)</b></td>
 
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      <td><b>Buffer (&mu;l)</b></td>
 
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      <td><b>T4 Ligase (&mu;l)</b></td>
 
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  </tr>
 
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    <tr>
 
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      <td><b>E1</b></td>
 
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      <td></html><partinfo>BBa_B0015</partinfo><html> (E-X)</td>
 
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      <td>1.5</td>
 
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      <td></html><partinfo>BBa_C0060</partinfo><html> (E-S)</td>
 
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      <td>6.5</td>
 
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      <td>1</td>
 
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      <td>1</td>
 
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  </tr>
 
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    <tr>
 
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      <td><b>E2</b></td>
 
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      <td></html><partinfo>BBa_B0030</partinfo><html> (S-P)</td>
 
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      <td>1.5</td>
 
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      <td></html><partinfo>BBa_C0061</partinfo><html> (X-P)</td>
 
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      <td>6.5</td>
 
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      <td>1</td>
 
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      <td>1</td>
 
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  </tr>
 
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    <tr>
 
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      <td><b>E3</b></td>
 
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      <td></html><partinfo>BBa_B0031</partinfo><html> (S-P)</td>
 
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      <td>1.5</td>
 
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      <td></html><partinfo>BBa_C0061</partinfo><html> (X-P)</td>
 
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      <td>6.5</td>
 
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      <td>1</td>
 
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      <td>1</td>
 
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  </tr>
 
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    <tr>
 
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      <td><b>E4</b></td>
 
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      <td></html><partinfo>BBa_B0032</partinfo><html> (S-P)</td>
 
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      <td>1.5</td>
 
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      <td></html><partinfo>BBa_C0061</partinfo><html> (X-P)</td>
 
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      <td>6.5</td>
 
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      <td>1</td>
 
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      <td>1</td>
 
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  </tr>
 
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    <tr>
 
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      <td><b>E8</b></td>
 
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      <td></html><partinfo>pSB4C5</partinfo><html> (E-P)</td>
 
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      <td>1.5</td>
 
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      <td></html><partinfo>BBa_K081022</partinfo><html> (E-P)</td>
 
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      <td>6.5</td>
 
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      <td>1</td>
 
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      <td>1</td>
 
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  </tr>
 
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</table>
 
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</center>
 
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<p>
 
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Ligations were incubated ON at 16°C.
 
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</p>
 
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<div align="right"><small><a href="#indice" title="">^top</a></small></div>
 
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</p>
 
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<a name="July.2C_19th"></a><h2> <span class="mw-headline">July, 19th</span></h2>
 
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<p>
 

Revision as of 00:48, 19 July 2011

UNIPV TEAM 2011

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JULY: WEEK 3

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Laboratory for Biomedical Informatics | Centre of Tissutal Engineering

University of Pavia

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