Difference between revisions of "Team:Pasteur Paris/Week 12"

Line 102: Line 102:
 
               <p></p><br><br><br>
 
               <p></p><br><br><br>
 
                
 
                
−
<h3 style="text-align:left"><em>Gibson  Assembly</em></h3><br />
+
<h3>NB-Esterase Assay:</h3>
−
<ul style="text-align:left">
+
<p>MiniPrep 808030 in the plasmid pDG011.</p>
−
<li>Gel  Migration of the PCR products (Cluster 2 and BBa_808014)</li>
+
<br />
−
<li>PCR  Amplification of intersequence 936011/936023 using Takara Ex Taq Polymerase.</li>
+
<h3>pNP-Assay</h3>
−
<li>Gel  Migration on 0,8% Agarose Gel</li>
+
<ul>
−
<li>PCR  Amplification of BBa_316003 using Takara Ex Taq Polymerase.</li>
+
<li>Culture of DH5-alpha transformed with 808030 in pDG011</li>
−
<li>Gel  Migration using 0,8% Agarose Gel</li>
+
<li>Enzymatic essay (pNP assay) of the first transformation (of the 11/08/15): BBa_808030 and pDG011 plasmid in DH5α</li>
−
<li>Spe I and  Pst I Restriction Digest of the recieved plasmids containing BBa_K936011,  BBa_K13932932, K936023.</li>
+
−
<li>Gel Purification of the digested  plasmids K936011 and K1392932 using the QIAgen Gel extraction kit protocol. </li>
+
 
</ul>
 
</ul>
−
 
+
<br/>
−
<br>
+
<h3>Gibson Assembly:</h3>
−
 
+
     <p>OD measurement of Cluster 4:</p>
−
<h3 style="text-align:left"><em>NB-Esterase assay </em></h3>
+
−
 
+
−
<ul style="text-align:left">
+
−
<li>Liquid culture of  the second transformation (of the 15/08/15): BBa_808030 and pDG011 plasmid in  DH5α </li>
+
−
<li>MiniPrep of  BBa_808030 in the plasmid pDG011 in BAP 1</li>
+
−
<li>XbaI and PstI digestion  of BBa_808030 and pDG011 (with the phosphatase step)</li>
+
−
</ul>
+
−
 
+
−
<br>
+
−
 
+
−
<p style="text-align:left"><em>Gel Purification</em></p>
+
−
 
+
−
<p style="text-align:left"><u>Tubes components:</u></p>
+
−
<table border="1" cellspacing="0" cellpadding="0" width="480">
+
−
  <tr>
+
−
     <td width="102" valign="top"><br />
+
−
      Tubes </td>
+
−
    <td width="217" valign="top"><p align="center">Ladder </p></td>
+
−
    <td width="161" valign="top"><p align="center">1st transformation </p></td>
+
−
  </tr>
+
−
  <tr>
+
−
    <td width="102" valign="top"><p>&nbsp;</p>
+
−
      <p>Components </p></td>
+
−
    <td width="217" valign="top"><p align="center"><strong>&nbsp;</strong></p>
+
−
      <p align="center"><strong>GeneRuler 1kb DNA Ladder</strong><br />
+
−
        (6x DNA    loading dye)<br />
+
−
        3µL</p></td>
+
−
    <td width="161" valign="top"><p align="center"><strong>Buffer</strong><br />
+
−
      4µL<br />
+
−
      +<br />
+
−
      <strong>DNA</strong> <br />
+
−
      20µL</p></td>
+
−
  </tr>
+
−
</table>
+
−
 
+
−
<br>
+
−
 
+
−
<ul style="text-align:left">
+
−
  <li>Ligation of BBa_808030 and pDG011 </li>
+
−
  <li>Transformation in Chemically Competent DH5-⍺ cells </li>
+
−
 
+
−
<li>Transformation  in DH5-ɑ of the biobrick K808030 in pDG011 </li>
+
−
<li>Transformation in DH5α cells of BBa_808030 insert and pDG011 vector </li>
+
−
 
+
−
 
+
−
  <li>SpeI and Pst  I Enzymatic Digest of BBa_K808030 with dephosphorylation.</li>
+
−
  <li>Gel migration</li>
+
−
  <li>Gel  extraction of the PCR product of K808030 and pDG011.</li>
+
−
  <li>Ligation of  pDG011 and K808030.</li>
+
−
</ul>
+
−
 
+
−
<br>
+
−
 
+
−
<p style="text-align:left">The bacteria did not grow  because the wrong antibiotic was chosen: BBa_J61047 was in pSB1A2 and not  pSB1C3.<br />
+
−
  - Bacterial culture  of BBa_J61047 on liquid LB + Amp 1X at 180 rpm, 37°C. The bacteria grew well. <br />
+
−
  - Midi-prep has been  done with the 50mL of BBa_J61047 culture. (dilution 1/1000) </p>
+
−
<table border="1" cellspacing="0" cellpadding="0" width="604">
+
−
  <tr>
+
−
    <td width="121" valign="top"><p>&nbsp;</p></td>
+
−
    <td width="121" valign="top"><p>DNA Concentration (ng/µl)</p></td>
+
−
    <td width="121" valign="top"><p>OD(260nm)</p></td>
+
−
    <td width="121" valign="top"><p>OD(280nm)</p></td>
+
−
    <td width="121" valign="top"><p>OD(260nm)/OD(280nm)</p></td>
+
−
  </tr>
+
−
  <tr>
+
−
    <td width="121" valign="top"><p>BBa_J61047</p></td>
+
−
    <td width="121" valign="top"><p align="right">0,5 </p></td>
+
−
    <td width="121" valign="top"><p align="right">0,011 </p></td>
+
−
    <td width="121" valign="top"><p align="right">0,004 </p></td>
+
−
    <td width="121" valign="top"><p align="right">2,57 </p></td>
+
−
  </tr>
+
−
</table>
+
−
<ul style="text-align:left"> <li>Preparation of YPD  specific media on specific media (1%  yeast extract, 2% Dextrose, 2% Peptone, 2% agar).</li>
+
−
 
+
−
  <li>Gel Migration of BBa_J61047.</li>
+
−
  <li>NotI enzymatic digest of BBa_J61047 in pSB1A2 enzyme. </li>
+
−
  <li>Gel Migration on a 0,8%  Agarose Gel and the digestion hasn&rsquo;t work. </li>
+
−
  <li>XbaI and SpeI digestion of BBa_J61047. </li>
+
−
  </ul>
+
−
 
+
−
<br>
+
−
 
+
−
  <ul style="text-align:left">
+
−
    <li>tube 1 «&nbsp;Miniprep XbaI/SpeI&nbsp;»&nbsp;:&nbsp;5uL 10X buffer cutsmart + 0,5uL  XbaI + 0,5uL SpeI + 38uL H2O +5uL ADN, digestion at 37°C during 1 hour.</li>
+
−
    <li>tube 2 «&nbsp;Midiprep XbaI/SpeI&nbsp;»&nbsp;: 5uL 10X buffer cut smart + 0,5uL XbaI +  0,5uL SpeI + 38uL H2O + 5uL ADN, digestion at 37°C during 1 hour</li>
+
−
    <li>tube 3 «&nbsp;Miniprep NotI&nbsp;»&nbsp;: 5uL 10X buffer cutsmart + 0,5 NotI +  38,5 H2O + 5uL ADN, digestion at 37°C during 1 hour</li>
+
−
    <li>tube 4 «&nbsp;MiDiprep NotI&nbsp;»&nbsp;: 5uL 10X buffer cutsmart + 0,5 NotI +  38,5 H2O + 5uL ADN, digestion at 37°C during 1 hour</li>
+
−
    <li>tube 5 «&nbsp;Miniprep CTRL&nbsp;» as negative  control&nbsp;: 5Ul ADN + 40uL H2O + 5uL 10X buffer cutsmart.</li>
+
−
    <li>tube 6 «&nbsp;Midiprep CTRL&nbsp;» as negative  control&nbsp;: 5Ul ADN + 40uL H2O + 5uL 10X buffer cutsmart.</li>
+
−
    <li>MWT&nbsp;: «&nbsp;Quick-Load Purple 2-Log DNA  Ladder (0.1-10.0 kb) NEB </li>
+
−
</ul>
+
−
 
+
−
</br>
+
−
 
+
−
<ul style="text-align:left">
+
−
  <li>PCR Amplification of the  BBa_J61047 using Phusion Polymerase Master Mix. </li>
+
−
  <li>Gel Migration on a 0.8% agarose gel (See figure 01)<br />
+
−
  </li>
+
−
  <li>Culture of the plasmid pRS415 in SC_Leu at 30°C at 150rpm for about 72h. </li>
+
−
  <li>PCR Amplification using the Phusion Polymerase of pRS415. </li>
+
−
</ul>
+
−
 
+
−
<br>
+
−
 
+
−
<p style="text-align:left"><em>NB-Esterase Assay: </em></p>
+
−
<ul style="text-align:left>
+
−
<li>MiniPrep 808030 in the plasmid pDG011</li>
+
−
<li>Gibson Assembly</li>
+
−
<li>OD measurement of Cluster 4</li>
+
−
</ul>
+
−
 
+
−
<div align="center">
+
 
   <table border="1" cellspacing="0" cellpadding="0" width="651">
 
   <table border="1" cellspacing="0" cellpadding="0" width="651">
 
     <tr>
 
     <tr>
 
       <td width="121" valign="top"><p>&nbsp;</p></td>
 
       <td width="121" valign="top"><p>&nbsp;</p></td>
−
       <td width="99" valign="top"><p align="center">Concentration (ng/µL) </p></td>
+
       <td width="99" valign="top"><p align="center">Concentration (ng/µL)</p></td>
−
       <td width="83" valign="top"><p align="center">OD(230nm) </p></td>
+
       <td width="83" valign="top"><p align="center">OD(230nm)</p></td>
−
       <td width="83" valign="top"><p align="center">OD(260nm) </p></td>
+
       <td width="83" valign="top"><p align="center">OD(260nm)</p></td>
−
       <td width="83" valign="top"><p align="center">OD(280nm) </p></td>
+
       <td width="83" valign="top"><p align="center">OD(280nm)</p></td>
−
       <td width="91" valign="top"><p align="center">OD(260nm)/OD(230nm) </p></td>
+
       <td width="91" valign="top"><p align="center">OD(260nm)/OD(230nm)</p></td>
−
       <td width="91" valign="top"><p align="center">OD(260nm)/OD(280nm) </p></td>
+
       <td width="91" valign="top"><p align="center">OD(260nm)/OD(280nm)</p></td>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
−
       <td width="121" valign="top"><p align="center">Cluster_4_start </p></td>
+
       <td width="121" valign="top"><p align="center">Cluster_4_start</p></td>
−
       <td width="99" valign="top"><p align="center">30 </p></td>
+
       <td width="99" valign="top"><p align="center">30</p></td>
−
       <td width="83" valign="top"><p align="center">0,025 </p></td>
+
       <td width="83" valign="top"><p align="center">0,025</p></td>
−
       <td width="83" valign="top"><p align="center">0,575 </p></td>
+
       <td width="83" valign="top"><p align="center">0,575</p></td>
−
       <td width="83" valign="top"><p align="center">0,375 </p></td>
+
       <td width="83" valign="top"><p align="center">0,375</p></td>
−
       <td width="91" valign="top"><p align="center">1,50 </p></td>
+
       <td width="91" valign="top"><p align="center">1,50</p></td>
−
       <td width="91" valign="top"><p align="center">- </p></td>
+
       <td width="91" valign="top"><p align="center">-</p></td>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
−
       <td width="121" valign="top"><p align="center">/936011 </p></td>
+
       <td width="121" valign="top"><p align="center">/936011</p></td>
−
       <td width="99" valign="top"><p align="center">55 </p></td>
+
       <td width="99" valign="top"><p align="center">55</p></td>
−
       <td width="83" valign="top"><p align="center">0,4 </p></td>
+
       <td width="83" valign="top"><p align="center">0,4</p></td>
−
       <td width="83" valign="top"><p align="center">1,075 </p></td>
+
       <td width="83" valign="top"><p align="center">1,075</p></td>
−
       <td width="83" valign="top"><p align="center">0,575 </p></td>
+
       <td width="83" valign="top"><p align="center">0,575</p></td>
−
       <td width="91" valign="top"><p align="center">1,88 </p></td>
+
       <td width="91" valign="top"><p align="center">1,88</p></td>
−
       <td width="91" valign="top"><p align="center">2,65 </p></td>
+
       <td width="91" valign="top"><p align="center">2,65</p></td>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
−
       <td width="121" valign="top"><p align="center">936011/936023 </p></td>
+
       <td width="121" valign="top"><p align="center">936011/936023</p></td>
−
       <td width="99" valign="top"><p align="center">7,5 </p></td>
+
       <td width="99" valign="top"><p align="center">7,5</p></td>
−
       <td width="83" valign="top"><p align="center">0,6 </p></td>
+
       <td width="83" valign="top"><p align="center">0,6</p></td>
−
       <td width="83" valign="top"><p align="center">0,125 </p></td>
+
       <td width="83" valign="top"><p align="center">0,125</p></td>
−
       <td width="83" valign="top"><p align="center">0,05 </p></td>
+
       <td width="83" valign="top"><p align="center">0,05</p></td>
−
       <td width="91" valign="top"><p align="center">2,41 </p></td>
+
       <td width="91" valign="top"><p align="center">2,41</p></td>
−
       <td width="91" valign="top"><p align="center">0,21 </p></td>
+
       <td width="91" valign="top"><p align="center">0,21</p></td>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
−
       <td width="121" valign="top"><p align="center">936023/ </p></td>
+
       <td width="121" valign="top"><p align="center">936023/</p></td>
−
       <td width="99" valign="top"><p align="center">45 </p></td>
+
       <td width="99" valign="top"><p align="center">45</p></td>
−
       <td width="83" valign="top"><p align="center">0,575 </p></td>
+
       <td width="83" valign="top"><p align="center">0,575</p></td>
−
       <td width="83" valign="top"><p align="center">0,925 </p></td>
+
       <td width="83" valign="top"><p align="center">0,925</p></td>
−
       <td width="83" valign="top"><p align="center">0,625 </p></td>
+
       <td width="83" valign="top"><p align="center">0,625</p></td>
−
       <td width="91" valign="top"><p align="center">1,48 </p></td>
+
       <td width="91" valign="top"><p align="center">1,48</p></td>
−
       <td width="91" valign="top"><p align="center">1,61 </p></td>
+
       <td width="91" valign="top"><p align="center">1,61</p></td>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
−
       <td width="121" valign="top"><p align="center">Cluster_4_end </p></td>
+
       <td width="121" valign="top"><p align="center">Cluster_4_end</p></td>
−
       <td width="99" valign="top"><p align="center">47,5 </p></td>
+
       <td width="99" valign="top"><p align="center">47,5</p></td>
−
       <td width="83" valign="top"><p align="center">0 </p></td>
+
       <td width="83" valign="top"><p align="center">0</p></td>
−
       <td width="83" valign="top"><p align="center">0,925 </p></td>
+
       <td width="83" valign="top"><p align="center">0,925</p></td>
−
       <td width="83" valign="top"><p align="center">0,575 </p></td>
+
       <td width="83" valign="top"><p align="center">0,575</p></td>
−
       <td width="91" valign="top"><p align="center">1,64 </p></td>
+
       <td width="91" valign="top"><p align="center">1,64</p></td>
−
       <td width="91" valign="top"><p align="center">- </p></td>
+
       <td width="91" valign="top"><p align="center">-</p></td>
 
     </tr>
 
     </tr>
 
   </table>
 
   </table>
−
</div>
+
  <br />
−
 
+
  <ul><li>Gel migration of the PCR product of prom/pNB Est and Cluster_1_start.</li></ul>
−
<p style="text-align:left">Gel plan:</p>
+
  <br />
−
<div align="center">
+
    <p>Gel plan:</p>
 +
   
 
   <table border="1" cellspacing="0" cellpadding="0" width="692">
 
   <table border="1" cellspacing="0" cellpadding="0" width="692">
 
     <tr>
 
     <tr>
−
       <td width="103" valign="top"><br />
+
       <td width="103" valign="top"><p align="center">Stain</p></td>
−
        Stain </td>
+
       <td width="53" valign="top"><p align="center">1</p></td>
−
       <td width="53" valign="top"><p align="center">1 </p></td>
+
       <td width="54" valign="top"><p align="center">2</p></td>
−
       <td width="54" valign="top"><p align="center">2 </p></td>
+
       <td width="53" valign="top"><p align="center">3</p></td>
−
       <td width="53" valign="top"><p align="center">3 </p></td>
+
       <td width="54" valign="top"><p align="center">4</p></td>
−
       <td width="54" valign="top"><p align="center">4 </p></td>
+
       <td width="53" valign="top"><p align="center">5</p></td>
−
       <td width="53" valign="top"><p align="center">5 </p></td>
+
       <td width="54" valign="top"><p align="center">6</p></td>
−
       <td width="54" valign="top"><p align="center">6 </p></td>
+
       <td width="53" valign="top"><p align="center">7</p></td>
−
       <td width="53" valign="top"><p align="center">7 </p></td>
+
       <td width="54" valign="top"><p align="center">8</p></td>
−
       <td width="54" valign="top"><p align="center">8 </p></td>
+
       <td width="53" valign="top"><p align="center">9</p></td>
−
       <td width="53" valign="top"><p align="center">9 </p></td>
+
       <td width="54" valign="top"><p align="center">10</p></td>
−
       <td width="54" valign="top"><p align="center">10 </p></td>
+
       <td width="54" valign="top"><p align="center">11</p></td>
−
       <td width="54" valign="top"><p align="center">11 </p></td>
+
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
−
       <td width="103" valign="top"><p align="center">Tube number </p></td>
+
       <td width="103" valign="top"><p align="center">Tube number</p></td>
 
       <td width="53" valign="top"><p>&nbsp;</p></td>
 
       <td width="53" valign="top"><p>&nbsp;</p></td>
 
       <td width="54" valign="top"><p>&nbsp;</p></td>
 
       <td width="54" valign="top"><p>&nbsp;</p></td>
−
       <td width="53" valign="top"><p align="center">A1 </p></td>
+
       <td width="53" valign="top"><p align="center">A1</p></td>
−
       <td width="54" valign="top"><p align="center">A2 </p></td>
+
       <td width="54" valign="top"><p align="center">A2</p></td>
−
       <td width="53" valign="top"><p align="center">A3 </p></td>
+
       <td width="53" valign="top"><p align="center">A3</p></td>
−
       <td width="54" valign="top"><p align="center">A4 </p></td>
+
       <td width="54" valign="top"><p align="center">A4</p></td>
 
       <td width="53" valign="top"><p>&nbsp;</p></td>
 
       <td width="53" valign="top"><p>&nbsp;</p></td>
−
       <td width="54" valign="top"><p align="center">B1 </p></td>
+
       <td width="54" valign="top"><p align="center">B1</p></td>
−
       <td width="53" valign="top"><p align="center">B2 </p></td>
+
       <td width="53" valign="top"><p align="center">B2</p></td>
−
       <td width="54" valign="top"><p align="center">B3 </p></td>
+
       <td width="54" valign="top"><p align="center">B3</p></td>
−
       <td width="54" valign="top"><p align="center">B4 </p></td>
+
       <td width="54" valign="top"><p align="center">B4</p></td>
 
     </tr>
 
     </tr>
 
     <tr>
 
     <tr>
−
       <td width="103" valign="top"><p align="center">Componants </p></td>
+
       <td width="103" valign="top"><p align="center">Componants</p></td>
−
       <td width="53" valign="top"><p align="center">Ladder </p></td>
+
       <td width="53" valign="top"><p align="center">Ladder</p></td>
 
       <td width="54" valign="top"><p>&nbsp;</p></td>
 
       <td width="54" valign="top"><p>&nbsp;</p></td>
 
       <td width="53" valign="top"><p>&nbsp;</p></td>
 
       <td width="53" valign="top"><p>&nbsp;</p></td>
Line 332: Line 218:
 
     </tr>
 
     </tr>
 
   </table>
 
   </table>
−
</div>
+
  <p><br/>
 +
   
 +
    <br/>
 +
    <p>DNA concentration assay :</p>
 +
   
 +
  <table border="1" cellspacing="0" cellpadding="0" width="678">
 +
    <tr>
 +
      <td width="127" valign="top"><p>&nbsp;</p></td>
 +
      <td width="100" valign="top"><p align="center">Concentration (ng/µL)</p></td>
 +
      <td width="83" valign="top"><p align="center">OD(230nm)</p></td>
 +
      <td width="92" valign="top"><p align="center">OD(260nm)</p></td>
 +
      <td width="92" valign="top"><p align="center">OD(280nm)</p></td>
 +
      <td width="92" valign="top"><p align="center">OD(260nm)/OD(280nm)</p></td>
 +
      <td width="92" valign="top"><p align="center">OD(260nm)/OD(230nm)</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="127" valign="top"><p>Cluster 4 start</p></td>
 +
      <td width="100" valign="top"><p>105</p></td>
 +
      <td width="83" valign="top"><p>0,3</p></td>
 +
      <td width="92" valign="top"><p>2,1</p></td>
 +
      <td width="92" valign="top"><p>1,1</p></td>
 +
      <td width="92" valign="top"><p>1,79</p></td>
 +
      <td width="92" valign="top"><p>2,40</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="127" valign="top"><p>/936011</p></td>
 +
      <td width="100" valign="top"><p>55</p></td>
 +
      <td width="83" valign="top"><p>0</p></td>
 +
      <td width="92" valign="top"><p>1,1</p></td>
 +
      <td width="92" valign="top"><p>0,55</p></td>
 +
      <td width="92" valign="top"><p>1,99</p></td>
 +
      <td width="92" valign="top"><p>-</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="127" valign="top"><p>936011/936023</p></td>
 +
      <td width="100" valign="top"><p>520</p></td>
 +
      <td width="83" valign="top"><p>4,325</p></td>
 +
      <td width="92" valign="top"><p>10,425</p></td>
 +
      <td width="92" valign="top"><p>5,825</p></td>
 +
      <td width="92" valign="top"><p>1,79</p></td>
 +
      <td width="92" valign="top"><p>4,38</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="127" valign="top"><p>936023/</p></td>
 +
      <td width="100" valign="top"><p>47,5</p></td>
 +
      <td width="83" valign="top"><p>0</p></td>
 +
      <td width="92" valign="top"><p>0,925</p></td>
 +
      <td width="92" valign="top"><p>0,5</p></td>
 +
      <td width="92" valign="top"><p>1,89</p></td>
 +
      <td width="92" valign="top"><p>-</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="127" valign="top"><p>Cluster 4 end</p></td>
 +
      <td width="100" valign="top"><p>130</p></td>
 +
      <td width="83" valign="top"><p>0,6</p></td>
 +
      <td width="92" valign="top"><p>2,625</p></td>
 +
      <td width="92" valign="top"><p>1,475</p></td>
 +
      <td width="92" valign="top"><p>1,79</p></td>
 +
      <td width="92" valign="top"><p>4,38</p></td>
 +
    </tr>
 +
  </table>
 +
<br/>
  
−
<br>
+
−
 
+
    <h3>Gibson Assembly:</h3>
−
<table border="1" cellspacing="0" cellpadding="0" width="678">
+
    <ul>
 +
    <li>Enzymatic digest of K936011, K936023 and K1392932</li>
 +
    <li>Enzymatic digest using Pst I and Spe I</li>
 +
    <li>Gel Migration of digested plasmids K936011, K936023, K1392932. on a 0,8% Agarose Gel</li>
 +
    <li>Gel Purification</li>
 +
    </ul>
 +
    <br />
 +
   
 +
    <strong>Cluster 4A Gibson assembly:</strong>
 +
    <ul>
 +
    <li>Gibson Assembly</li>
 +
    <li>PCR amplification of cluster 4A Assembly.</li>
 +
    <li>Gel migration on 0,8% Agarose Gel.</li>
 +
    </ul>
 +
    <br/>
 +
    <p>Gel Plan:</p>
 +
   
 +
  <table border="1" cellspacing="0" cellpadding="0" width="642">
 +
    <tr>
 +
      <td width="107" valign="top"><p>Well    Number</p></td>
 +
      <td width="107" valign="top"><p align="right">1</p></td>
 +
      <td width="107" valign="top"><p align="right">2</p></td>
 +
      <td width="107" valign="top"><p align="right">3</p></td>
 +
      <td width="107" valign="top"><p align="right">4</p></td>
 +
      <td width="107" valign="top"><p align="right">5</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="107" valign="top"><p>Contains</p></td>
 +
      <td width="107" valign="top"><p>DNA    ladder</p></td>
 +
      <td width="107" valign="top"><p>No    primers</p></td>
 +
      <td width="107" valign="top"><p>Primer    for only</p></td>
 +
      <td width="107" valign="top"><p>Primer    rev only</p></td>
 +
      <td width="107" valign="top"><p>Both    primers</p></td>
 +
    </tr>
 +
  </table>
 +
    <br/>
 +
    <h3>PCR amplification of Biobricks K808007, K808030, K936011, K936023, K1392932, K316003</h3>
 +
    <ul>
 +
    <li>PCR amplification using Phusion polymerase</li>
 +
    <li>Gel Migration on a 1% Agarose Gel</li>
 +
    </ul>
 +
    <br/>
 +
    <p>
 +
    The PCR worked for most of our Biobricks with the exception of the biobrick K808030.</p>
 +
    <br/>
 +
    <p>DNA concentration measurement</p>
 +
  <table border="1" cellspacing="0" cellpadding="0" width="605">
 +
    <tr>
 +
      <td width="64" valign="top"><p>1/25    dilution</p></td>
 +
      <td width="77" valign="top"><p>DNA    concentration (ng/µL)</p></td>
 +
      <td width="77" valign="top"><p>OD(230nm)</p></td>
 +
      <td width="77" valign="top"><p>OD(260nm)</p></td>
 +
      <td width="77" valign="top"><p>OD(280nm)</p></td>
 +
      <td width="77" valign="top"><p>OD(340nm)</p></td>
 +
      <td width="77" valign="top"><p>OD(260nm)/OD(280nm)</p></td>
 +
      <td width="77" valign="top"><p>OD(260nm)/OD(230nm)</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="64" valign="top"><p align="right">808007</p></td>
 +
      <td width="77" valign="top"><p align="right">4,3</p></td>
 +
      <td width="77" valign="top"><p align="right">0,08</p></td>
 +
      <td width="77" valign="top"><p align="right">0,085</p></td>
 +
      <td width="77" valign="top"><p align="right">0,052</p></td>
 +
      <td width="77" valign="top"><p align="right">0,006</p></td>
 +
      <td width="77" valign="top"><p align="right">1,64</p></td>
 +
      <td width="77" valign="top"><p align="right">1,06</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="64" valign="top"><p align="right">808030</p></td>
 +
      <td width="77" valign="top"><p align="right">4,6</p></td>
 +
      <td width="77" valign="top"><p align="right">0,151</p></td>
 +
      <td width="77" valign="top"><p align="right">0,092</p></td>
 +
      <td width="77" valign="top"><p align="right">0,058</p></td>
 +
      <td width="77" valign="top"><p align="right">0,003</p></td>
 +
      <td width="77" valign="top"><p align="right">1,59</p></td>
 +
      <td width="77" valign="top"><p align="right">1,61</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="64" valign="top"><p align="right">936011</p></td>
 +
      <td width="77" valign="top"><p align="right">5,9</p></td>
 +
      <td width="77" valign="top"><p align="right">0,141</p></td>
 +
      <td width="77" valign="top"><p align="right">0,118</p></td>
 +
      <td width="77" valign="top"><p align="right">0,073</p></td>
 +
      <td width="77" valign="top"><p align="right">0,009</p></td>
 +
      <td width="77" valign="top"><p align="right">1,61</p></td>
 +
      <td width="77" valign="top"><p align="right">0,83</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="64" valign="top"><p align="right">936023</p></td>
 +
      <td width="77" valign="top"><p align="right">5,2</p></td>
 +
      <td width="77" valign="top"><p align="right">0,097</p></td>
 +
      <td width="77" valign="top"><p align="right">0,105</p></td>
 +
      <td width="77" valign="top"><p align="right">0,062</p></td>
 +
      <td width="77" valign="top"><p align="right">0,003</p></td>
 +
      <td width="77" valign="top"><p align="right">1,7</p></td>
 +
      <td width="77" valign="top"><p align="right">1,08</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="64" valign="top"><p align="right">1392932</p></td>
 +
      <td width="77" valign="top"><p align="right">12,6</p></td>
 +
      <td width="77" valign="top"><p align="right">0,493</p></td>
 +
      <td width="77" valign="top"><p align="right">0,251</p></td>
 +
      <td width="77" valign="top"><p align="right">0,259</p></td>
 +
      <td width="77" valign="top"><p align="right">0,035</p></td>
 +
      <td width="77" valign="top"><p align="right">0,97</p></td>
 +
      <td width="77" valign="top"><p align="right">0,51</p></td>
 +
    </tr>
 +
    <tr>
 +
      <td width="64" valign="top"><p align="right">316003</p></td>
 +
      <td width="77" valign="top"><p align="right">5,5</p></td>
 +
      <td width="77" valign="top"><p align="right">0,1</p></td>
 +
      <td width="77" valign="top"><p align="right">0,11</p></td>
 +
      <td width="77" valign="top"><p align="right">0,062</p></td>
 +
      <td width="77" valign="top"><p align="right">0,0</p></td>
 +
      <td width="77" valign="top"><p align="right">1,76</p></td>
 +
      <td width="77" valign="top"><p align="right">1,10</p></td>
 +
    </tr>
 +
  </table>
 +
<br />
 +
      <h3>PCR amplification of pSB1C3</h3>
 +
    <ul>
 +
    <li>PCR amplification using Phusion Polymerase.</li>
 +
    <li>Gel Migration on a 1% agarose gel</li>
 +
    </ul>
 +
<table border="1" cellspacing="0" cellpadding="0" width="642">
 
   <tr>
 
   <tr>
−
     <td width="127" valign="top"><p>&nbsp;</p></td>
+
     <td width="80" valign="top"><p>Well    Number</p></td>
−
     <td width="100" valign="top"><p align="center">Concentration (ng/µL) </p></td>
+
     <td width="80" valign="top"><p align="right">1</p></td>
−
     <td width="83" valign="top"><p align="center">OD(230nm)</p></td>
+
     <td width="80" valign="top"><p align="right">2</p></td>
−
     <td width="92" valign="top"><p align="center">OD(260nm)</p></td>
+
     <td width="80" valign="top"><p align="right">3</p></td>
−
     <td width="92" valign="top"><p align="center">OD(280nm)</p></td>
+
     <td width="80" valign="top"><p align="right">4</p></td>
−
     <td width="92" valign="top"><p align="center">OD(260nm)/OD(280nm)</p></td>
+
     <td width="80" valign="top"><p align="right">5</p></td>
−
     <td width="92" valign="top"><p align="center">OD(260nm)/OD(230nm)</p></td>
+
     <td width="80" valign="top"><p align="right">6</p></td>
 +
    <td width="80" valign="top"><p align="right">7</p></td>
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
−
     <td width="127" valign="top"><p>Cluster 4 start </p></td>
+
     <td width="80" valign="top"><p>Contains</p></td>
−
     <td width="100" valign="top"><p>105 </p></td>
+
     <td width="80" valign="top"><p>DNA    ladder</p></td>
−
     <td width="83" valign="top"><p>0,3 </p></td>
+
     <td width="80" valign="top"><p>Non    digested Plasmid</p></td>
−
     <td width="92" valign="top"><p>2,1 </p></td>
+
     <td width="80" valign="top"><p>Digested    plasmid</p></td>
−
     <td width="92" valign="top"><p>1,1 </p></td>
+
     <td width="80" valign="top"><p>No    primers</p></td>
−
     <td width="92" valign="top"><p>1,79 </p></td>
+
     <td width="80" valign="top"><p>Primer    for only</p></td>
−
     <td width="92" valign="top"><p>2,40 </p></td>
+
     <td width="80" valign="top"><p>Primer    rev only</p></td>
 +
    <td width="80" valign="top"><p>Both    primers</p></td>
 
   </tr>
 
   </tr>
 +
</table>
 +
    <br/>
 +
    <h3>PCR amplification of pSB1C3</h3>
 +
    <ul>
 +
    <li>PCR amplification using Takara Ex Taq Polymerase testing different annealing temperatures.</li>
 +
    </ul>
 +
<table border="1" cellspacing="0" cellpadding="0" width="400">
 
   <tr>
 
   <tr>
−
     <td width="127" valign="top"><p>/936011 </p></td>
+
     <td width="133" valign="top"><p>&nbsp;</p></td>
−
    <td width="100" valign="top"><p>55 </p></td>
+
     <td width="77" valign="top"><p>No    primers</p></td>
−
    <td width="83" valign="top"><p>0 </p></td>
+
     <td width="71" valign="top"><p>Primer    for</p></td>
−
     <td width="92" valign="top"><p>1,1 </p></td>
+
     <td width="73" valign="top"><p>Primer    rev</p></td>
−
     <td width="92" valign="top"><p>0,55 </p></td>
+
     <td width="45" valign="top"><p>&nbsp;</p></td>
−
     <td width="92" valign="top"><p>1,99 </p></td>
+
−
     <td width="92" valign="top"><p>- </p></td>
+
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
−
     <td width="127" valign="top"><p>936011/936023 </p></td>
+
     <td width="133" valign="top"><p>DNAse,    RNAse free water</p></td>
−
     <td width="100" valign="top"><p>520 </p></td>
+
     <td width="77" valign="top"><p align="right">41,75</p></td>
−
    <td width="83" valign="top"><p>4,325 </p></td>
+
     <td width="71" valign="top"><p align="right">40,75</p></td>
−
     <td width="92" valign="top"><p>10,425 </p></td>
+
     <td width="73" valign="top"><p align="right">40,75</p></td>
−
     <td width="92" valign="top"><p>5,825 </p></td>
+
     <td width="45" valign="top"><p align="right">39,75</p></td>
−
     <td width="92" valign="top"><p>1,79 </p></td>
+
−
    <td width="92" valign="top"><p>4,38 </p></td>
+
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
−
     <td width="127" valign="top"><p>936023/ </p></td>
+
     <td width="133" valign="top"><p>10X    Ex Taq Buffer</p></td>
−
     <td width="100" valign="top"><p>47,5 </p></td>
+
     <td width="77" valign="top"><p align="right">5</p></td>
−
     <td width="83" valign="top"><p>0 </p></td>
+
     <td width="71" valign="top"><p align="right">5</p></td>
−
     <td width="92" valign="top"><p>0,925 </p></td>
+
     <td width="73" valign="top"><p align="right">5</p></td>
−
    <td width="92" valign="top"><p>0,5 </p></td>
+
     <td width="45" valign="top"><p align="right">5</p></td>
−
     <td width="92" valign="top"><p>1,89 </p></td>
+
−
    <td width="92" valign="top"><p>- </p></td>
+
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
−
     <td width="127" valign="top"><p>Cluster 4 end </p></td>
+
     <td width="133" valign="top"><p>dNTP</p></td>
−
     <td width="100" valign="top"><p>130 </p></td>
+
     <td width="77" valign="top"><p align="right">2</p></td>
−
     <td width="83" valign="top"><p>0,6 </p></td>
+
     <td width="71" valign="top"><p align="right">2</p></td>
−
     <td width="92" valign="top"><p>2,625 </p></td>
+
     <td width="73" valign="top"><p align="right">2</p></td>
−
     <td width="92" valign="top"><p>1,475 </p></td>
+
     <td width="45" valign="top"><p align="right">2</p></td>
−
     <td width="92" valign="top"><p>1,79 </p></td>
+
  </tr>
−
     <td width="92" valign="top"><p>4,38 </p></td>
+
  <tr>
 +
    <td width="133" valign="top"><p>rescue_for</p></td>
 +
    <td width="77" valign="top"><p align="right">0</p></td>
 +
    <td width="71" valign="top"><p align="right">1</p></td>
 +
     <td width="73" valign="top"><p align="right">0</p></td>
 +
    <td width="45" valign="top"><p align="right">1</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="133" valign="top"><p>rescue_rev </p></td>
 +
    <td width="77" valign="top"><p align="right">0</p></td>
 +
    <td width="71" valign="top"><p align="right">0</p></td>
 +
    <td width="73" valign="top"><p align="right">1</p></td>
 +
    <td width="45" valign="top"><p align="right">1</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="133" valign="top"><p>DNA  Template</p></td>
 +
    <td width="77" valign="top"><p align="right">1</p></td>
 +
    <td width="71" valign="top"><p align="right">1</p></td>
 +
    <td width="73" valign="top"><p align="right">1</p></td>
 +
    <td width="45" valign="top"><p align="right">1</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="133" valign="top"><p>Ex    Taq Polymerase</p></td>
 +
    <td width="77" valign="top"><p align="right">0,25</p></td>
 +
     <td width="71" valign="top"><p align="right">0,25</p></td>
 +
    <td width="73" valign="top"><p align="right">0,25</p></td>
 +
    <td width="45" valign="top"><p align="right">0,25</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="133" valign="top"><p>Total</p></td>
 +
    <td width="77" valign="top"><p align="right">50</p></td>
 +
    <td width="71" valign="top"><p align="right">50</p></td>
 +
    <td width="73" valign="top"><p align="right">50</p></td>
 +
    <td width="45" valign="top"><p align="right">50</p></td>
 
   </tr>
 
   </tr>
 
</table>
 
</table>
 +
<br/>
 +
  <P> Cycles:</p>
 +
  <ul>
 +
    <li>95°C for 4min</li>
 +
    <li>25 cycles :</li>
 +
    <li>95°C for 40s</li>
 +
    <li>annealing for 30s:</li>
 +
    <li>60°C: Tube 1-4</li>
 +
    <li>61,1°C: Tube 5-8</li>
 +
    <li>63°C: Tube 9-12</li>
 +
    <li>65,6°C: Tube 13-16</li>
 +
    <li>69,2°C: Tube 17-20</li>
 +
    <li>72,1°C: Tube 21-24</li>
 +
    <li>73,9°C: Tube 25-28</li>
 +
    <li>75°C: Tube 28-32</li>
 +
    <li>72°C for 80s</li>
 +
    <li>72°C for 7min.</li>
 +
<br/>
 +
<br/>
 +
<h3>PCR amplification using Takara Ex Taq</h3>
 +
    <ul>
 +
    <li>Gel Migration on a 1% Agarose Gel.</li>
 +
    </ul>
  
−
<br>
+
    <h3>PCR amplification of Biobricks K808030</h3>
−
<p style="text-align:left"><u>DNA  concentration assay</u></p>
+
    <ul>
−
<br>
+
    <li>PCR Amplification using Takara Ex Taq Polymerase.</li>
−
<p style="text-align:left">pNP-Assay </p>
+
   <li>Gel migration on 1%</li>
−
<ol style="text-align:left">
+
   </ul>
−
   <li><u>Culture of DH5-alpha transformed with 808030 in pDG011</u></li>
+
  <br/>
−
   <li><u>Enzymatic essay (<strong>pNP assay</strong>) of the first transformation (of the  11/08/15): BBa_808030 and pDG011 plasmid in DH5</u><u>α</u></li>
+
  <p>Gel Plan:</p>
−
</ol>
+
  
−
<br>
 
−
<p style="text-align:left">Gibson  Assembly : <br />
 
−
  Enzymatic  digest of K936011, K936023 and K1392932 </p>
 
−
<ol style="text-align:left">
 
−
  <li>Enzymatic digest using Pst I and Spe I </li>
 
−
  <li>Gel Migration of digested plasmids K936011, K936023,  K1392932. on a 0,8% Agarose Gel </li>
 
−
  <li>Gel Purification </li>
 
−
</ol>
 
−
 
−
<br>
 
−
 
−
<p style="text-align:left"><strong>Cluster 4A  Gibson assembly</strong><strong> </strong>:</p>
 
−
<ol style="text-align:left">
 
−
  <li>Gibson Assembly </li>
 
−
  <li>PCR amplification of cluster 4A Assembly. </li>
 
−
  <li>Gel migration on 0,8% Agarose Gel. </li>
 
−
</ol>
 
−
 
−
<br>
 
−
 
−
<p style="text-align:left">Gel Plan: </p>
 
 
<table border="1" cellspacing="0" cellpadding="0" width="642">
 
<table border="1" cellspacing="0" cellpadding="0" width="642">
 
   <tr>
 
   <tr>
−
     <td width="107" valign="top"><br />
+
     <td width="107" valign="top"><p>Well    Number</p></td>
−
      Well    Number </td>
+
     <td width="107" valign="top"><p align="right">1</p></td>
−
     <td width="107" valign="top"><p align="right">1 </p></td>
+
     <td width="107" valign="top"><p align="right">2</p></td>
−
     <td width="107" valign="top"><p align="right">2 </p></td>
+
     <td width="107" valign="top"><p align="right">3</p></td>
−
     <td width="107" valign="top"><p align="right">3 </p></td>
+
     <td width="107" valign="top"><p align="right">4</p></td>
−
     <td width="107" valign="top"><p align="right">4 </p></td>
+
     <td width="107" valign="top"><p align="right">5</p></td>
−
     <td width="107" valign="top"><p align="right">5 </p></td>
+
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
−
     <td width="107" valign="top"><p>Contains</p></td>
+
     <td width="107" valign="top"><p>&nbsp;</p></td>
 
     <td width="107" valign="top"><p>DNA    ladder</p></td>
 
     <td width="107" valign="top"><p>DNA    ladder</p></td>
 
     <td width="107" valign="top"><p>No    primers</p></td>
 
     <td width="107" valign="top"><p>No    primers</p></td>
−
     <td width="107" valign="top"><p>Primer    for only</p></td>
+
     <td width="107" valign="top"><p>Primer    For</p></td>
−
     <td width="107" valign="top"><p>Primer    rev only</p></td>
+
     <td width="107" valign="top"><p>Primer    Rev</p></td>
−
     <td width="107" valign="top"><p>Both    primers</p></td>
+
     <td width="107" valign="top"><p>Extract</p></td>
 
   </tr>
 
   </tr>
 
</table>
 
</table>
−
 
+
<br />
−
<p style="text-align:left"><strong>Gel Migration&nbsp;on a 1% Agarose Gel</strong></p>
+
<br />
−
 
+
    <h3>PCR amplification of pSB1C3</h3>
−
<p style="text-align:left">As you can see, the PCR worked for most of our  Biobricks with the exception of the biobrick K808030. </p>
+
    <ul>
−
<ol style="text-align:left">
+
    <li>PCR amplification using Takara Ex Taq Polymerase testing different annealing temperatures.</li>
−
  <li><strong>DNA  concentration measurement</strong><strong> </strong></li>
+
    <li>Gel Migration on a 1% Agarose Gel.</li>
−
</ol>
+
    </ul>
−
<table border="1" cellspacing="0" cellpadding="0" width="605">
+
   
 +
    <h3>PCR amplification of pSB1C3</h3>
 +
    <ul>
 +
    <li>PCR amplification using Takara Ex Taq Polymerase testing 8 annealing temperatures.</li>
 +
    </ul>
 +
<table border="1" cellspacing="0" cellpadding="0" width="400">
 
   <tr>
 
   <tr>
−
     <td width="64" valign="top"><p>1/25    dilution</p></td>
+
     <td width="133" valign="top"><p>&nbsp;</p></td>
−
     <td width="77" valign="top"><p>DNA   concentration (ng/µL)</p></td>
+
     <td width="77" valign="top"><p>No   primers</p></td>
−
     <td width="77" valign="top"><p>OD(230nm)</p></td>
+
     <td width="71" valign="top"><p>Primer    for</p></td>
−
     <td width="77" valign="top"><p>OD(260nm)</p></td>
+
     <td width="73" valign="top"><p>Primer    rev</p></td>
−
     <td width="77" valign="top"><p>OD(280nm)</p></td>
+
     <td width="45" valign="top"><p>&nbsp;</p></td>
−
    <td width="77" valign="top"><p>OD(340nm)</p></td>
+
−
    <td width="77" valign="top"><p>OD(260nm)/OD(280nm)</p></td>
+
−
    <td width="77" valign="top"><p>OD(260nm)/OD(230nm)</p></td>
+
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
−
     <td width="64" valign="top"><p align="right">808007 </p></td>
+
     <td width="133" valign="top"><p>DNAse,   RNAse free water</p></td>
−
    <td width="77" valign="top"><p align="right">4,3 </p></td>
+
     <td width="77" valign="top"><p align="right">41,75</p></td>
−
     <td width="77" valign="top"><p align="right">0,08 </p></td>
+
     <td width="71" valign="top"><p align="right">40,75</p></td>
−
     <td width="77" valign="top"><p align="right">0,085 </p></td>
+
     <td width="73" valign="top"><p align="right">40,75</p></td>
−
     <td width="77" valign="top"><p align="right">0,052 </p></td>
+
     <td width="45" valign="top"><p align="right">39,75</p></td>
−
     <td width="77" valign="top"><p align="right">0,006 </p></td>
+
−
    <td width="77" valign="top"><p align="right">1,64 </p></td>
+
−
    <td width="77" valign="top"><p align="right">1,06 </p></td>
+
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
−
     <td width="64" valign="top"><p align="right">808030 </p></td>
+
     <td width="133" valign="top"><p>10X    Ex Taq Buffer</p></td>
−
     <td width="77" valign="top"><p align="right">4,6 </p></td>
+
     <td width="77" valign="top"><p align="right">5</p></td>
−
     <td width="77" valign="top"><p align="right">0,151 </p></td>
+
     <td width="71" valign="top"><p align="right">5</p></td>
−
     <td width="77" valign="top"><p align="right">0,092 </p></td>
+
     <td width="73" valign="top"><p align="right">5</p></td>
−
     <td width="77" valign="top"><p align="right">0,058 </p></td>
+
     <td width="45" valign="top"><p align="right">5</p></td>
−
    <td width="77" valign="top"><p align="right">0,003 </p></td>
+
−
    <td width="77" valign="top"><p align="right">1,59 </p></td>
+
−
    <td width="77" valign="top"><p align="right">1,61 </p></td>
+
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
−
     <td width="64" valign="top"><p align="right">936011 </p></td>
+
     <td width="133" valign="top"><p>dNTP</p></td>
−
     <td width="77" valign="top"><p align="right">5,9 </p></td>
+
     <td width="77" valign="top"><p align="right">2</p></td>
−
     <td width="77" valign="top"><p align="right">0,141 </p></td>
+
     <td width="71" valign="top"><p align="right">2</p></td>
−
     <td width="77" valign="top"><p align="right">0,118 </p></td>
+
     <td width="73" valign="top"><p align="right">2</p></td>
−
     <td width="77" valign="top"><p align="right">0,073 </p></td>
+
     <td width="45" valign="top"><p align="right">2</p></td>
−
    <td width="77" valign="top"><p align="right">0,009 </p></td>
+
−
    <td width="77" valign="top"><p align="right">1,61 </p></td>
+
−
    <td width="77" valign="top"><p align="right">0,83 </p></td>
+
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
−
     <td width="64" valign="top"><p align="right">936023 </p></td>
+
     <td width="133" valign="top"><p>rescue_for</p></td>
−
    <td width="77" valign="top"><p align="right">5,2 </p></td>
+
     <td width="77" valign="top"><p align="right">0</p></td>
−
     <td width="77" valign="top"><p align="right">0,097 </p></td>
+
     <td width="71" valign="top"><p align="right">1</p></td>
−
     <td width="77" valign="top"><p align="right">0,105 </p></td>
+
     <td width="73" valign="top"><p align="right">0</p></td>
−
     <td width="77" valign="top"><p align="right">0,062 </p></td>
+
     <td width="45" valign="top"><p align="right">1</p></td>
−
     <td width="77" valign="top"><p align="right">0,003 </p></td>
+
−
    <td width="77" valign="top"><p align="right">1,7 </p></td>
+
−
    <td width="77" valign="top"><p align="right">1,08 </p></td>
+
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
−
     <td width="64" valign="top"><p align="right">1392932 </p></td>
+
     <td width="133" valign="top"><p>rescue_rev </p></td>
−
    <td width="77" valign="top"><p align="right">12,6 </p></td>
+
     <td width="77" valign="top"><p align="right">0</p></td>
−
     <td width="77" valign="top"><p align="right">0,493 </p></td>
+
     <td width="71" valign="top"><p align="right">0</p></td>
−
     <td width="77" valign="top"><p align="right">0,251 </p></td>
+
     <td width="73" valign="top"><p align="right">1</p></td>
−
     <td width="77" valign="top"><p align="right">0,259 </p></td>
+
     <td width="45" valign="top"><p align="right">1</p></td>
−
     <td width="77" valign="top"><p align="right">0,035 </p></td>
+
−
    <td width="77" valign="top"><p align="right">0,97 </p></td>
+
−
    <td width="77" valign="top"><p align="right">0,51 </p></td>
+
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
−
     <td width="64" valign="top"><p align="right">316003 </p></td>
+
     <td width="133" valign="top"><p>DNA  Template</p></td>
−
     <td width="77" valign="top"><p align="right">5,5 </p></td>
+
     <td width="77" valign="top"><p align="right">1</p></td>
−
     <td width="77" valign="top"><p align="right">0,1 </p></td>
+
     <td width="71" valign="top"><p align="right">1</p></td>
−
     <td width="77" valign="top"><p align="right">0,11 </p></td>
+
     <td width="73" valign="top"><p align="right">1</p></td>
−
    <td width="77" valign="top"><p align="right">0,062 </p></td>
+
     <td width="45" valign="top"><p align="right">1</p></td>
−
    <td width="77" valign="top"><p align="right">0,0 </p></td>
+
−
    <td width="77" valign="top"><p align="right">1,76 </p></td>
+
−
     <td width="77" valign="top"><p align="right">1,10 </p></td>
+
 
   </tr>
 
   </tr>
−
</table>
 
−
 
−
<p style="text-align:left"><strong>PCR  amplification of pSB1C3</strong></p>
 
−
<ol style="text-align:left">
 
−
  <li><strong>PCR amplification</strong><strong> using Phusion Polymerase.</strong><strong> </strong></li>
 
−
  <li><strong>Gel  Migration on a 1% agarose gel</strong><strong> </strong></li>
 
−
</ol>
 
−
<table border="1" cellspacing="0" cellpadding="0" width="642">
 
 
   <tr>
 
   <tr>
−
     <td width="80" valign="top"><p>Well   Number</p></td>
+
     <td width="133" valign="top"><p>Ex   Taq Polymerase</p></td>
−
     <td width="80" valign="top"><p align="right">1 </p></td>
+
     <td width="77" valign="top"><p align="right">0,25</p></td>
−
     <td width="80" valign="top"><p align="right">2 </p></td>
+
     <td width="71" valign="top"><p align="right">0,25</p></td>
−
     <td width="80" valign="top"><p align="right">3 </p></td>
+
     <td width="73" valign="top"><p align="right">0,25</p></td>
−
     <td width="80" valign="top"><p align="right">4 </p></td>
+
     <td width="45" valign="top"><p align="right">0,25</p></td>
−
    <td width="80" valign="top"><p align="right">5 </p></td>
+
−
    <td width="80" valign="top"><p align="right">6 </p></td>
+
−
    <td width="80" valign="top"><p align="right">7 </p></td>
+
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
−
     <td width="80" valign="top"><p>Contains</p></td>
+
     <td width="133" valign="top"><p>Total</p></td>
−
     <td width="80" valign="top"><p>DNA    ladder</p></td>
+
     <td width="77" valign="top"><p align="right">50</p></td>
−
     <td width="80" valign="top"><p>Non    digested Plasmid</p></td>
+
     <td width="71" valign="top"><p align="right">50</p></td>
−
    <td width="80" valign="top"><p>Digested    plasmid</p></td>
+
     <td width="73" valign="top"><p align="right">50</p></td>
−
     <td width="80" valign="top"><p>No    primers</p></td>
+
     <td width="45" valign="top"><p align="right">50</p></td>
−
    <td width="80" valign="top"><p>Primer    for only</p></td>
+
−
     <td width="80" valign="top"><p>Primer    rev only</p></td>
+
−
    <td width="80" valign="top"><p>Both    primers</p></td>
+
 
   </tr>
 
   </tr>
 
</table>
 
</table>
−
<p>&nbsp;</p>
+
<br/>
−
<p style="text-align:left">PCR amplification of pSB1C3 <br />
+
      
−
  - PCR  amplification using Takara  Ex Taq Polymerase testing different annealing temperatures. </p>
+
     <h3>Gel Migration on a 1% Agarose Gel</h3>
−
<div align="center">
+
     <p>Enzymatic digest of BBa_K808030</p>
−
  <table border="1" cellspacing="0" cellpadding="0" width="400">
+
    <ul>
−
     <tr>
+
    <li>Enzymatic digest by XbaI and Spe I of BBa_K808030</li>
−
      <td width="133" valign="top"><p>&nbsp;</p></td>
+
    <li>Gel migration.</li>
−
      <td width="77" valign="top"><p>No    primers</p></td>
+
    </ul>
−
      <td width="71" valign="top"><p>Primer    for</p></td>
+
<br />
−
      <td width="73" valign="top"><p>Primer    rev</p></td>
+
    <h3>Yeast Assembly</h3>
−
      <td width="45" valign="top"><p>&nbsp;</p></td>
+
    <p>The bacteria did not grow because the wrong antibiotic was chosen: BBa_J61047 was in pSB1A2 and not pSB1C3.</p>
−
     </tr>
+
    <ul>
−
    <tr>
+
    <li>Bacterial culture of BBa_J61047 on liquid LB + Amp 1X at 180 rpm, 37°C. The bacteria grew well.</li>
−
      <td width="133" valign="top"><p>DNAse,    RNAse free water</p></td>
+
    <li>Midi-prep has been done with the 50mL of BBa_J61047 culture. (dilution 1/1000)</li>
−
      <td width="77" valign="top"><p align="right">41,75 </p></td>
+
    </ul>
−
      <td width="71" valign="top"><p align="right">40,75 </p></td>
+
−
      <td width="73" valign="top"><p align="right">40,75 </p></td>
+
−
      <td width="45" valign="top"><p align="right">39,75 </p></td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td width="133" valign="top"><p>10X    Ex Taq Buffer</p></td>
+
−
      <td width="77" valign="top"><p align="right">5 </p></td>
+
−
      <td width="71" valign="top"><p align="right">5 </p></td>
+
−
      <td width="73" valign="top"><p align="right">5 </p></td>
+
−
      <td width="45" valign="top"><p align="right">5 </p></td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td width="133" valign="top"><p>dNTP</p></td>
+
−
      <td width="77" valign="top"><p align="right">2 </p></td>
+
−
      <td width="71" valign="top"><p align="right">2 </p></td>
+
−
      <td width="73" valign="top"><p align="right">2 </p></td>
+
−
      <td width="45" valign="top"><p align="right">2 </p></td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td width="133" valign="top"><p>rescue_for</p></td>
+
−
      <td width="77" valign="top"><p align="right">0 </p></td>
+
−
      <td width="71" valign="top"><p align="right">1 </p></td>
+
−
      <td width="73" valign="top"><p align="right">0 </p></td>
+
−
      <td width="45" valign="top"><p align="right">1 </p></td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td width="133" valign="top"><p>rescue_rev </p></td>
+
−
      <td width="77" valign="top"><p align="right">0 </p></td>
+
−
      <td width="71" valign="top"><p align="right">0 </p></td>
+
−
      <td width="73" valign="top"><p align="right">1 </p></td>
+
−
      <td width="45" valign="top"><p align="right">1 </p></td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td width="133" valign="top"><p>DNA  Template</p></td>
+
−
      <td width="77" valign="top"><p align="right">1 </p></td>
+
−
      <td width="71" valign="top"><p align="right">1 </p></td>
+
−
      <td width="73" valign="top"><p align="right">1 </p></td>
+
−
      <td width="45" valign="top"><p align="right">1 </p></td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td width="133" valign="top"><p>Ex    Taq Polymerase</p></td>
+
−
      <td width="77" valign="top"><p align="right">0,25 </p></td>
+
−
      <td width="71" valign="top"><p align="right">0,25 </p></td>
+
−
      <td width="73" valign="top"><p align="right">0,25 </p></td>
+
−
      <td width="45" valign="top"><p align="right">0,25 </p></td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td width="133" valign="top"><p>Total</p></td>
+
−
      <td width="77" valign="top"><p align="right">50 </p></td>
+
−
      <td width="71" valign="top"><p align="right">50 </p></td>
+
−
      <td width="73" valign="top"><p align="right">50 </p></td>
+
−
      <td width="45" valign="top"><p align="right">50 </p></td>
+
−
    </tr>
+
−
  </table>
+
−
</div>
+
−
<p style="text-align:left">Cycles: </p>
+
−
<ol style="text-align:left">
+
−
  <li>95°C for 4min </li>
+
−
  <li>25 cycles : </li>
+
−
  <ol>
+
−
    <li>95°C for 40s </li>
+
−
    <li>annealing for 30s: </li>
+
−
    <ol>
+
−
      <li>60°C: Tube 1-4 </li>
+
−
      <li>61,1°C: Tube 5-8 </li>
+
−
      <li>63°C: Tube 9-12 </li>
+
−
      <li>65,6°C: Tube 13-16 </li>
+
−
      <li>69,2°C: Tube 17-20 </li>
+
−
      <li>72,1°C: Tube 21-24 </li>
+
−
      <li>73,9°C: Tube 25-28 </li>
+
−
      <li>75°C: Tube 28-32 </li>
+
−
    </ol>
+
−
    <li>72°C for 80s </li>
+
−
  </ol>
+
−
  <li>72°C for 7min. </li>
+
−
</ol>
+
−
 
+
−
<p style="text-align:left"><strong>PCR  amplification of Biobricks K808030</strong><strong> </strong></p>
+
−
<ol style="text-align:left">
+
−
  <li><strong>PCR  Amplification using Takara Ex Taq Polymerase.</strong><strong> </strong></li>
+
−
  <li><strong>Gel migration on 1%</strong><strong> </strong></li>
+
−
</ol>
+
−
<p style="text-align:left">Gel Plan: </p>
+
−
<table border="1" cellspacing="0" cellpadding="0" width="642">
+
−
  <tr>
+
−
    <td width="107" valign="top"><br />
+
−
      Well    Number </td>
+
−
    <td width="107" valign="top"><p align="right">1 </p></td>
+
−
    <td width="107" valign="top"><p align="right">2 </p></td>
+
−
    <td width="107" valign="top"><p align="right">3 </p></td>
+
−
    <td width="107" valign="top"><p align="right">4 </p></td>
+
−
    <td width="107" valign="top"><p align="right">5 </p></td>
+
−
  </tr>
+
−
  <tr>
+
−
    <td width="107" valign="top"><p>&nbsp;</p></td>
+
−
    <td width="107" valign="top"><p>DNA    ladder</p></td>
+
−
    <td width="107" valign="top"><p>No    primers</p></td>
+
−
    <td width="107" valign="top"><p>Primer    For</p></td>
+
−
    <td width="107" valign="top"><p>Primer    Rev</p></td>
+
−
    <td width="107" valign="top"><p>Extract</p></td>
+
−
  </tr>
+
−
</table>
+
−
 
+
−
<p style="text-align:left"><strong>PCR  amplification of pSB1C</strong><strong>3</strong><strong> </strong></p>
+
−
<ol style="text-align:left">
+
−
  <li><strong>PCR amplification</strong><strong> using Takara Ex Taq Polymerase  testing different annealing temperatures.</strong><strong> </strong></li>
+
−
  <li><strong>Gel  Migration on a 1% Agarose Gel.</strong><strong> </strong></li>
+
−
</ol>
+
−
<p><strong>PCR  amplification of pSB1C3</strong><strong> </strong></p>
+
−
<ol style="text-align:left">
+
−
  <li><strong>PCR amplification using Takara Ex Taq Polymerase  testing 8 annealing temperatures.</strong></li>
+
−
</ol>
+
−
 
+
−
<div align="center">
+
−
  <table border="1" cellspacing="0" cellpadding="0" width="400">
+
−
     <tr>
+
−
      <td width="133" valign="top"><p>&nbsp;</p></td>
+
−
      <td width="77" valign="top"><p>No    primers</p></td>
+
−
      <td width="71" valign="top"><p>Primer    for</p></td>
+
−
      <td width="73" valign="top"><p>Primer    rev</p></td>
+
−
      <td width="45" valign="top"><p>&nbsp;</p></td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td width="133" valign="top"><p>DNAse,    RNAse free water</p></td>
+
−
      <td width="77" valign="top"><p align="right">41,75 </p></td>
+
−
      <td width="71" valign="top"><p align="right">40,75 </p></td>
+
−
      <td width="73" valign="top"><p align="right">40,75 </p></td>
+
−
      <td width="45" valign="top"><p align="right">39,75 </p></td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td width="133" valign="top"><p>10X    Ex Taq Buffer</p></td>
+
−
      <td width="77" valign="top"><p align="right">5 </p></td>
+
−
      <td width="71" valign="top"><p align="right">5 </p></td>
+
−
      <td width="73" valign="top"><p align="right">5 </p></td>
+
−
      <td width="45" valign="top"><p align="right">5 </p></td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td width="133" valign="top"><p>dNTP</p></td>
+
−
      <td width="77" valign="top"><p align="right">2 </p></td>
+
−
      <td width="71" valign="top"><p align="right">2 </p></td>
+
−
      <td width="73" valign="top"><p align="right">2 </p></td>
+
−
      <td width="45" valign="top"><p align="right">2 </p></td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td width="133" valign="top"><p>rescue_for</p></td>
+
−
      <td width="77" valign="top"><p align="right">0 </p></td>
+
−
      <td width="71" valign="top"><p align="right">1 </p></td>
+
−
      <td width="73" valign="top"><p align="right">0 </p></td>
+
−
      <td width="45" valign="top"><p align="right">1 </p></td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td width="133" valign="top"><p>rescue_rev </p></td>
+
−
      <td width="77" valign="top"><p align="right">0 </p></td>
+
−
      <td width="71" valign="top"><p align="right">0 </p></td>
+
−
      <td width="73" valign="top"><p align="right">1 </p></td>
+
−
      <td width="45" valign="top"><p align="right">1 </p></td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td width="133" valign="top"><p>DNA  Template</p></td>
+
−
      <td width="77" valign="top"><p align="right">1 </p></td>
+
−
      <td width="71" valign="top"><p align="right">1 </p></td>
+
−
      <td width="73" valign="top"><p align="right">1 </p></td>
+
−
      <td width="45" valign="top"><p align="right">1 </p></td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td width="133" valign="top"><p>Ex    Taq Polymerase</p></td>
+
−
      <td width="77" valign="top"><p align="right">0,25 </p></td>
+
−
      <td width="71" valign="top"><p align="right">0,25 </p></td>
+
−
      <td width="73" valign="top"><p align="right">0,25 </p></td>
+
−
      <td width="45" valign="top"><p align="right">0,25 </p></td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td width="133" valign="top"><p>Total</p></td>
+
−
      <td width="77" valign="top"><p align="right">50 </p></td>
+
−
      <td width="71" valign="top"><p align="right">50 </p></td>
+
−
      <td width="73" valign="top"><p align="right">50 </p></td>
+
−
      <td width="45" valign="top"><p align="right">50 </p></td>
+
−
    </tr>
+
−
  </table>
+
−
</div>
+
−
<p style="text-align:left">Cycles: </p>
+
−
<ol style="text-align:left">
+
−
  <li>95°C for 4min </li>
+
−
  <li>25 cycles : </li>
+
−
  <ol>
+
−
    <li>95°C for 40s </li>
+
−
    <li>annealing for 30s: </li>
+
−
    <ol>
+
−
      <li>60°C: Tube 1-4 </li>
+
−
      <li>61,1°C: Tube 5-8 </li>
+
−
      <li>63°C: Tube 9-12 </li>
+
−
      <li>65,6°C: Tube 13-16 </li>
+
−
      <li>69,2°C: Tube 17-20 </li>
+
−
      <li>72,1°C: Tube 21-24 </li>
+
−
      <li>73,9°C: Tube 25-28 </li>
+
−
      <li>75°C: Tube 28-32 </li>
+
−
    </ol>
+
−
    <li>72°C for 80s </li>
+
−
  </ol>
+
−
  <li>72°C for 7min. </li>
+
−
</ol>
+
−
<p style="text-align:left"><strong>2) Gel  Migration on a 1% Agarose Gel</strong><strong> </strong><br />
+
−
</p>
+
−
<p style="text-align:left">Enzymatic digest of BBa_K808030 </p>
+
−
<ol style="text-align:left">
+
−
  <li>Enzymatic digest by XbaI and Spe I of BBa_K808030 </li>
+
−
  <li>Gel migration. </li>
+
−
</ol>
+
−
<p style="text-align:left">Yeast Assembly :<br />
+
−
  The bacteria did not grow because the wrong antibiotic was chosen: BBa_J61047 was in pSB1A2 and not pSB1C3.<br />
+
−
  Bacterial culture of BBa_J61047 on liquid LB + Amp 1X at 180 rpm, 37°C. The bacteria grew well. <br />
+
−
Midi-prep has been done with the 50mL of BBa_J61047 culture. (dilution 1/1000) </p>
+
 
<table border="1" cellspacing="0" cellpadding="0" width="604">
 
<table border="1" cellspacing="0" cellpadding="0" width="604">
 
   <tr>
 
   <tr>
Line 786: Line 651:
 
   <tr>
 
   <tr>
 
     <td width="121" valign="top"><p>BBa_J61047</p></td>
 
     <td width="121" valign="top"><p>BBa_J61047</p></td>
−
     <td width="121" valign="top"><p align="right">0,5 </p></td>
+
     <td width="121" valign="top"><p align="right">0,5</p></td>
−
     <td width="121" valign="top"><p align="right">0,011 </p></td>
+
     <td width="121" valign="top"><p align="right">0,011</p></td>
−
     <td width="121" valign="top"><p align="right">0,004 </p></td>
+
     <td width="121" valign="top"><p align="right">0,004</p></td>
−
     <td width="121" valign="top"><p align="right">2,57 </p></td>
+
     <td width="121" valign="top"><p align="right">2,57</p></td>
 
   </tr>
 
   </tr>
 
</table>
 
</table>
−
<p style="text-align:left"><u>
+
<br/>
−
</u>Preparation of YPD specific media on specific media (1% yeast extract, 2% Dextrose, 2% Peptone, 2% agar).</p>
+
    <ul>
−
<ol style="text-align:left">
+
    <li> Preparation of YPD specific media on specific media (1% yeast extract, 2% Dextrose, 2% Peptone, 2% agar).</li>
−
  <li>Gel Migration of BBa_J61047.<u> </u></li>
+
    <li>Gel Migration of BBa_J61047.</li>  
−
  <li>NotI enzymatic digest of BBa_J61047 in pSB1A2 enzyme. </li>
+
    <li>NotI enzymatic digest of BBa_J61047 in pSB1A2 enzyme.</li>
−
  <li>Gel Migration on a 0,8% Agarose Gel and the digestion hasn&rsquo;t work. </li>
+
    <li>Gel Migration on a 0,8% Agarose Gel and the digestion hasn&rsquo;t work.</li>
−
  <li>XbaI and SpeI digestion of BBa_J61047. <br />
+
    <li>XbaI and SpeI digestion of BBa_J61047.</li>
−
     <br />
+
    </ul>
−
</ol>
+
   
 +
    <ul>
 +
    <li>tube 1 « Miniprep XbaI/SpeI » : 5uL 10X buffer cutsmart + 0,5uL XbaI + 0,5uL SpeI + 38uL H2O +5uL ADN, digestion at 37°C during 1 hour.</li>
 +
    <li>tube 2 « Midiprep XbaI/SpeI » : 5uL 10X buffer cut smart + 0,5uL XbaI + 0,5uL SpeI + 38uL H2O + 5uL ADN, digestion at 37°C during 1 hour</li>
 +
    <li>tube 3 « Miniprep NotI » : 5uL 10X buffer cutsmart + 0,5 NotI + 38,5 H2O + 5uL ADN, digestion at 37°C during 1 hour</li>
 +
    <li>tube 4 « MiDiprep NotI » : 5uL 10X buffer cutsmart + 0,5 NotI + 38,5 H2O + 5uL ADN, digestion at 37°C during 1 hour</li>
 +
    <li>tube 5 « Miniprep CTRL » as negative control : 5Ul ADN + 40uL H2O + 5uL 10X buffer cutsmart.</li>
 +
    <li>tube 6 « Midiprep CTRL » as negative control : 5Ul ADN + 40uL H2O + 5uL 10X buffer cutsmart.</li>
 +
    <li>MWT : « Quick-Load Purple 2-Log DNA Ladder (0.1-10.0 kb) NEB</li>
 +
    <br/>
 +
     <br/>
 +
 
 +
    <h3>PCR Amplification of the BBa_J61047 using Phusion Polymerase Master Mix.</h3>
 +
    <ul>
 +
    <li>Gel Migration on a 0.8% agarose gel</li>
 +
    <li>Culture of the plasmid pRS415 in SC_Leu at 30°C at 150rpm for about 72h.</li>
 +
    <li>PCR Amplification using the Phusion Polymerase of pRS415.</li>
 +
    <li>Gel Extraction of the PCR products. </li>
 +
    <br/>
 +
    <br/>
 +
    <h3>TPA solubility :</h3>
 +
    <ul>
 +
    <li>Determination of the solubilization products of TPA.</li>
 +
    <li>Detection of 2-hydroxy-terephthalate using a TECAN spectrometer.</li>
 +
    <li>Precipitaion of terephthalic acid in H2SO4.</li>
  
−
<ul style="text-align:left">
 
−
    <li>tube 1 «&nbsp;Miniprep XbaI/SpeI&nbsp;»&nbsp;:&nbsp;5uL 10X buffer cutsmart + 0,5uL  XbaI + 0,5uL SpeI + 38uL H2O +5uL ADN, digestion at 37°C during 1 hour.</li>
 
−
    <li>tube 2 «&nbsp;Midiprep XbaI/SpeI&nbsp;»&nbsp;: 5uL 10X buffer cut smart + 0,5uL XbaI +  0,5uL SpeI + 38uL H2O + 5uL ADN, digestion at 37°C during 1 hour.</li>
 
−
    <li> tube 3 «&nbsp;Miniprep NotI&nbsp;»&nbsp;: 5uL 10X buffer cutsmart + 0,5 NotI +  38,5 H2O + 5uL ADN, digestion at 37°C during 1 hour</li>
 
−
    <li>tube 4 «&nbsp;MiDiprep NotI&nbsp;»&nbsp;: 5uL 10X buffer cutsmart + 0,5 NotI +  38,5 H2O + 5uL ADN, digestion at 37°C during 1 hour</li>
 
−
    <li>tube 5 «&nbsp;Miniprep CTRL&nbsp;» as negative  control&nbsp;: 5Ul ADN + 40uL H2O + 5uL 10X buffer cutsmart.</li>
 
−
    <li>tube 6 «&nbsp;Midiprep CTRL&nbsp;» as negative  control&nbsp;: 5Ul ADN + 40uL H2O + 5uL 10X buffer cutsmart.</li>
 
−
    <li>MWT&nbsp;: «&nbsp;Quick-Load Purple 2-Log DNA  Ladder (0.1-10.0 kb) NEB </li>
 
−
</ul>
 
  
−
<ol style="text-align:left">
 
−
  <li>PCR Amplification of the  BBa_J61047 using Phusion Polymerase Master Mix. </li>
 
−
  <li>Gel Migration on a 0.8% agarose gel (See figure 01)<br />
 
−
  </li>
 
−
  <li>Culture of the plasmid pRS415 in SC_Leu at 30°C at 150rpm for about 72h. </li>
 
−
  <li>PCR Amplification using the Phusion Polymerase of pRS415. </li>
 
−
</ol>
 
−
<p style="text-align:left">Gel Extraction of the PCR  products.&nbsp;</p>
 
  
−
<p style="text-align:left"><em>TPA solubility</em></p>
 
−
<ol style="text-align:left">
 
−
  <li>Determination of the  solubilization products of TPA. </li>
 
−
  <ol>
 
−
    <li>Detection of  2-hydroxy-terephthalate using a TECAN spectrometer. </li>
 
−
  </ol>
 
−
  <li>Precipitaion of terephthalic  acid in H2SO4:</li>
 
−
 
 
−
</ol>
 
−
</ol>
 
  
 
<br>
 
<br>

Revision as of 00:14, 19 September 2015

Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

Week 7

Week 8

Week 9

Week 10

Week 11

Week 12

Week 13

Week 14

Week 15

Week 12

08/17 - 08/21




NB-Esterase Assay:

MiniPrep 808030 in the plasmid pDG011.


pNP-Assay

  • Culture of DH5-alpha transformed with 808030 in pDG011
  • Enzymatic essay (pNP assay) of the first transformation (of the 11/08/15): BBa_808030 and pDG011 plasmid in DH5α

Gibson Assembly:

OD measurement of Cluster 4:

 

Concentration (ng/µL)

OD(230nm)

OD(260nm)

OD(280nm)

OD(260nm)/OD(230nm)

OD(260nm)/OD(280nm)

Cluster_4_start

30

0,025

0,575

0,375

1,50

-

/936011

55

0,4

1,075

0,575

1,88

2,65

936011/936023

7,5

0,6

0,125

0,05

2,41

0,21

936023/

45

0,575

0,925

0,625

1,48

1,61

Cluster_4_end

47,5

0

0,925

0,575

1,64

-

  • Gel migration of the PCR product of prom/pNB Est and Cluster_1_start.

Gel plan:

Stain

1

2

3

4

5

6

7

8

9

10

11

Tube number

 

 

A1

A2

A3

A4

 

B1

B2

B3

B4

Componants

Ladder

 

 

 

 

 

 

 

 

 

 



DNA concentration assay :

 

Concentration (ng/µL)

OD(230nm)

OD(260nm)

OD(280nm)

OD(260nm)/OD(280nm)

OD(260nm)/OD(230nm)

Cluster 4 start

105

0,3

2,1

1,1

1,79

2,40

/936011

55

0

1,1

0,55

1,99

-

936011/936023

520

4,325

10,425

5,825

1,79

4,38

936023/

47,5

0

0,925

0,5

1,89

-

Cluster 4 end

130

0,6

2,625

1,475

1,79

4,38

Gibson Assembly:

  • Enzymatic digest of K936011, K936023 and K1392932
  • Enzymatic digest using Pst I and Spe I
  • Gel Migration of digested plasmids K936011, K936023, K1392932. on a 0,8% Agarose Gel
  • Gel Purification

Cluster 4A Gibson assembly:
  • Gibson Assembly
  • PCR amplification of cluster 4A Assembly.
  • Gel migration on 0,8% Agarose Gel.

Gel Plan:

Well Number

1

2

3

4

5

Contains

DNA ladder

No primers

Primer for only

Primer rev only

Both primers

PCR amplification of Biobricks K808007, K808030, K936011, K936023, K1392932, K316003

  • PCR amplification using Phusion polymerase
  • Gel Migration on a 1% Agarose Gel

The PCR worked for most of our Biobricks with the exception of the biobrick K808030.


DNA concentration measurement

1/25 dilution

DNA concentration (ng/µL)

OD(230nm)

OD(260nm)

OD(280nm)

OD(340nm)

OD(260nm)/OD(280nm)

OD(260nm)/OD(230nm)

808007

4,3

0,08

0,085

0,052

0,006

1,64

1,06

808030

4,6

0,151

0,092

0,058

0,003

1,59

1,61

936011

5,9

0,141

0,118

0,073

0,009

1,61

0,83

936023

5,2

0,097

0,105

0,062

0,003

1,7

1,08

1392932

12,6

0,493

0,251

0,259

0,035

0,97

0,51

316003

5,5

0,1

0,11

0,062

0,0

1,76

1,10

PCR amplification of pSB1C3

  • PCR amplification using Phusion Polymerase.
  • Gel Migration on a 1% agarose gel

Well Number

1

2

3

4

5

6

7

Contains

DNA ladder

Non digested Plasmid

Digested plasmid

No primers

Primer for only

Primer rev only

Both primers

PCR amplification of pSB1C3

  • PCR amplification using Takara Ex Taq Polymerase testing different annealing temperatures.

 

No primers

Primer for

Primer rev

 

DNAse, RNAse free water

41,75

40,75

40,75

39,75

10X Ex Taq Buffer

5

5

5

5

dNTP

2

2

2

2

rescue_for

0

1

0

1

rescue_rev

0

0

1

1

DNA  Template

1

1

1

1

Ex Taq Polymerase

0,25

0,25

0,25

0,25

Total

50

50

50

50

Cycles:

  • 95°C for 4min
  • 25 cycles :
  • 95°C for 40s
  • annealing for 30s:
  • 60°C: Tube 1-4
  • 61,1°C: Tube 5-8
  • 63°C: Tube 9-12
  • 65,6°C: Tube 13-16
  • 69,2°C: Tube 17-20
  • 72,1°C: Tube 21-24
  • 73,9°C: Tube 25-28
  • 75°C: Tube 28-32
  • 72°C for 80s
  • 72°C for 7min.


    • PCR amplification using Takara Ex Taq

    • Gel Migration on a 1% Agarose Gel.

    PCR amplification of Biobricks K808030

    • PCR Amplification using Takara Ex Taq Polymerase.
    • Gel migration on 1%

    Gel Plan:

    Well Number

    1

    2

    3

    4

    5

     

    DNA ladder

    No primers

    Primer For

    Primer Rev

    Extract


    PCR amplification of pSB1C3

    • PCR amplification using Takara Ex Taq Polymerase testing different annealing temperatures.
    • Gel Migration on a 1% Agarose Gel.

    PCR amplification of pSB1C3

    • PCR amplification using Takara Ex Taq Polymerase testing 8 annealing temperatures.

     

    No primers

    Primer for

    Primer rev

     

    DNAse, RNAse free water

    41,75

    40,75

    40,75

    39,75

    10X Ex Taq Buffer

    5

    5

    5

    5

    dNTP

    2

    2

    2

    2

    rescue_for

    0

    1

    0

    1

    rescue_rev

    0

    0

    1

    1

    DNA  Template

    1

    1

    1

    1

    Ex Taq Polymerase

    0,25

    0,25

    0,25

    0,25

    Total

    50

    50

    50

    50

    Gel Migration on a 1% Agarose Gel

    Enzymatic digest of BBa_K808030

    • Enzymatic digest by XbaI and Spe I of BBa_K808030
    • Gel migration.

    Yeast Assembly

    The bacteria did not grow because the wrong antibiotic was chosen: BBa_J61047 was in pSB1A2 and not pSB1C3.

    • Bacterial culture of BBa_J61047 on liquid LB + Amp 1X at 180 rpm, 37°C. The bacteria grew well.
    • Midi-prep has been done with the 50mL of BBa_J61047 culture. (dilution 1/1000)

     

    DNA Concentration (ng/µl)

    OD(260nm)

    OD(280nm)

    OD(260nm)/OD(280nm)

    BBa_J61047

    0,5

    0,011

    0,004

    2,57

    • Preparation of YPD specific media on specific media (1% yeast extract, 2% Dextrose, 2% Peptone, 2% agar).
    • Gel Migration of BBa_J61047.
    • NotI enzymatic digest of BBa_J61047 in pSB1A2 enzyme.
    • Gel Migration on a 0,8% Agarose Gel and the digestion hasn’t work.
    • XbaI and SpeI digestion of BBa_J61047.
    • tube 1 « Miniprep XbaI/SpeI » : 5uL 10X buffer cutsmart + 0,5uL XbaI + 0,5uL SpeI + 38uL H2O +5uL ADN, digestion at 37°C during 1 hour.
    • tube 2 « Midiprep XbaI/SpeI » : 5uL 10X buffer cut smart + 0,5uL XbaI + 0,5uL SpeI + 38uL H2O + 5uL ADN, digestion at 37°C during 1 hour
    • tube 3 « Miniprep NotI » : 5uL 10X buffer cutsmart + 0,5 NotI + 38,5 H2O + 5uL ADN, digestion at 37°C during 1 hour
    • tube 4 « MiDiprep NotI » : 5uL 10X buffer cutsmart + 0,5 NotI + 38,5 H2O + 5uL ADN, digestion at 37°C during 1 hour
    • tube 5 « Miniprep CTRL » as negative control : 5Ul ADN + 40uL H2O + 5uL 10X buffer cutsmart.
    • tube 6 « Midiprep CTRL » as negative control : 5Ul ADN + 40uL H2O + 5uL 10X buffer cutsmart.
    • MWT : « Quick-Load Purple 2-Log DNA Ladder (0.1-10.0 kb) NEB


      • PCR Amplification of the BBa_J61047 using Phusion Polymerase Master Mix.

      • Gel Migration on a 0.8% agarose gel
      • Culture of the plasmid pRS415 in SC_Leu at 30°C at 150rpm for about 72h.
      • PCR Amplification using the Phusion Polymerase of pRS415.
      • Gel Extraction of the PCR products. 


        • TPA solubility :

        • Determination of the solubilization products of TPA.
        • Detection of 2-hydroxy-terephthalate using a TECAN spectrometer.
        • Precipitaion of terephthalic acid in H2SO4.


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