Difference between revisions of "Team:Pasteur Paris/Week 12"
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− | <td style="vertical-align: top; width: | + | <td style="vertical-align: top; width: 90%; overflow: scroll;" class="fontcorpspage"> |
<p><span class="titrepage">Week 12</span><br/><br/> | <p><span class="titrepage">Week 12</span><br/><br/> | ||
+ | |||
<span class="soustitrepage">08/17 - 08/21</span></p> | <span class="soustitrepage">08/17 - 08/21</span></p> | ||
− | |||
− | + | <p></p><br> | |
+ | |||
+ | <center><h3><em> NB-Esterase Assay </em></h3></center></br> | ||
+ | <ul style="text-align:left"> | ||
+ | <li>MiniPrep 808030 in the plasmid pDG011.</li> | ||
+ | </ul></br> | ||
+ | |||
+ | <center><h3><em>pNP-Assay</em></h3></center></br> | ||
+ | <ul style="text-align:left"> | ||
+ | <li>Culture of DH5-α transformed with 808030 in pDG011</li> | ||
+ | <li>Enzymatic assay (pNP assay) of the first transformation (of the 11/08/15): BBa_808030 and pDG011 plasmid in DH5-α</li> | ||
+ | </ul> | ||
+ | <br/> | ||
+ | <center><h3><em>Gibson Assembly</em></h3></center></br> | ||
+ | <p style="text-align:left">OD measurement of Cluster 4:</p> | ||
+ | <table border="1" cellspacing="0" cellpadding="0" width="651"> | ||
+ | <tr> | ||
+ | <td width="121" valign="top"><p> </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(260nm)</p></td> | ||
+ | <td width="83" valign="top"><p align="center">OD(280nm)</p></td> | ||
+ | <td width="91" valign="top"><p align="center">OD(260/230nm)</p></td> | ||
+ | <td width="91" valign="top"><p align="center">OD(260/280nm)</p></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <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="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">0.375</p></td> | ||
+ | <td width="91" valign="top"><p align="center">1.50</p></td> | ||
+ | <td width="91" valign="top"><p align="center">-</p></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="121" valign="top"><p align="center">/936011</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">1.075</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">2.65</p></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <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="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">0.05</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> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="121" valign="top"><p align="center">936023/</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">0.925</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">1.61</p></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <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="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">0.575</p></td> | ||
+ | <td width="91" valign="top"><p align="center">1.64</p></td> | ||
+ | <td width="91" valign="top"><p align="center">-</p></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <br /> | ||
+ | <ul style="text-align:left"><li>Gel migration of the PCR product of prom/pNB Est and Cluster_1_start.</li></ul> | ||
+ | <br /> | ||
+ | <p style="text-align:left">Gel plan:</p> | ||
+ | |||
+ | <table border="1" cellspacing="0" cellpadding="0" width="692"> | ||
+ | <tr> | ||
+ | <td width="103" valign="top"><p align="center">Stain</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="53" valign="top"><p align="center">3</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="54" valign="top"><p align="center">6</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="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">11</p></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="103" valign="top"><p align="center">Tube number</p></td> | ||
+ | <td width="53" valign="top"><p> </p></td> | ||
+ | <td width="54" valign="top"><p> </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="53" valign="top"><p align="center">A3</p></td> | ||
+ | <td width="54" valign="top"><p align="center">A4</p></td> | ||
+ | <td width="53" valign="top"><p> </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="54" valign="top"><p align="center">B3</p></td> | ||
+ | <td width="54" valign="top"><p align="center">B4</p></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td width="103" valign="top"><p align="center">Componants</p></td> | ||
+ | <td width="53" valign="top"><p align="center">Ladder</p></td> | ||
+ | <td width="54" valign="top"><p> </p></td> | ||
+ | <td width="53" valign="top"><p> </p></td> | ||
+ | <td width="54" valign="top"><p> </p></td> | ||
+ | <td width="53" valign="top"><p> </p></td> | ||
+ | <td width="54" valign="top"><p> </p></td> | ||
+ | <td width="53" valign="top"><p> </p></td> | ||
+ | <td width="54" valign="top"><p> </p></td> | ||
+ | <td width="53" valign="top"><p> </p></td> | ||
+ | <td width="54" valign="top"><p> </p></td> | ||
+ | <td width="54" valign="top"><p> </p></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <br/> | ||
+ | |||
+ | <br/> | ||
+ | <center><h3><em>DNA concentration assay</em></h3></center></br> | ||
+ | |||
+ | <table border="1" cellspacing="0" cellpadding="0" width="678"> | ||
+ | <tr> | ||
+ | <td width="127" valign="top"><p> </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(260/280nm)</p></td> | ||
+ | <td width="92" valign="top"><p align="center">OD(260/230nm)</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/> | ||
+ | |||
+ | |||
+ | <center><h3><em>Gibson Assembly</em></h3></center></br> | ||
+ | <ul style="text-align:left"> | ||
+ | <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 /> | ||
+ | |||
+ | <center><h3><em>Cluster 4A Gibson assembly</em></h3></center></br> | ||
+ | <ul 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> | ||
+ | <li>Gel plan</em></li> | ||
+ | </ul> | ||
+ | </br> | ||
+ | |||
+ | <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/> | ||
+ | |||
+ | <center><h3><em>PCR amplification of Biobricks BBa_K808007, BBa_K808030, BBa_K936011, BBa_K936023, BBa_K1392932, BBa_K316003</em></h3></center></br> | ||
+ | <ul style="text-align:left"> | ||
+ | <li>PCR amplification using Phusion polymerase</li> | ||
+ | <li>Gel Migration on a 1% agarose gel</li> | ||
+ | </ul> | ||
+ | <br/> | ||
+ | <p style="text-align:left"> | ||
+ | The PCR worked for most of our Biobricks with the exception of the biobrick K808030.</p> | ||
+ | <br/> | ||
+ | <p style="text-align:left">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(260/280nm)</p></td> | ||
+ | <td width="77" valign="top"><p>OD(260/230nm)</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 /> | ||
+ | <center><h3><em>PCR amplification of pSB1C3</em></h3></center></br> | ||
+ | <ul style="text-align:left"> | ||
+ | <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> | ||
+ | <td width="80" valign="top"><p>Well Number</p></td> | ||
+ | <td width="80" valign="top"><p align="right">1</p></td> | ||
+ | <td width="80" valign="top"><p align="right">2</p></td> | ||
+ | <td width="80" valign="top"><p align="right">3</p></td> | ||
+ | <td width="80" valign="top"><p align="right">4</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> | ||
+ | <td width="80" valign="top"><p>Contains</p></td> | ||
+ | <td width="80" valign="top"><p>DNA ladder</p></td> | ||
+ | <td width="80" valign="top"><p>Non digested Plasmid</p></td> | ||
+ | <td width="80" valign="top"><p>Digested plasmid</p></td> | ||
+ | <td width="80" valign="top"><p>No primers</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> | ||
+ | </table> | ||
+ | <br/> | ||
+ | |||
+ | <center><h3><em>PCR amplification of pSB1C3</em></h3></center></br> | ||
+ | <ul style="text-align:left"> | ||
+ | <li>PCR amplification using Takara Ex Taq Polymerase testing different annealing temperatures.</li> | ||
+ | </ul> | ||
+ | <table border="1" cellspacing="0" cellpadding="0" width="400"> | ||
+ | <tr> | ||
+ | <td width="133" valign="top"><p> </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> </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> | ||
+ | <br/> | ||
+ | <p style="text-align:left"> Cycles:</p> | ||
+ | <ul style="text-align:left"> | ||
+ | <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 29-32</li> | ||
+ | <li>72°C for 80s</li> | ||
+ | <li>72°C for 7min.</li> | ||
+ | <br/> | ||
+ | <br/> | ||
+ | |||
+ | <center><h3><em>PCR amplification using Takara Ex Taq</em></h3></center></br> | ||
+ | <ul style="text-align:left"> | ||
+ | <li>Gel Migration on a 1% agarose gel.</li> | ||
+ | </ul> | ||
+ | |||
+ | <center><h3><em>PCR amplification of Biobricks K808030</em></h3></center></br> | ||
+ | <ul style="text-align:left"> | ||
+ | <li>PCR Amplification using Takara Ex Taq Polymerase.</li> | ||
+ | <li>Gel migration on 1%</li> | ||
+ | </ul> | ||
+ | <br/> | ||
+ | <p style="text-align:left">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> </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> | ||
+ | <br /> | ||
+ | <br /> | ||
+ | <center><h3><em>PCR amplification of pSB1C3</em></h3></center></br> | ||
+ | <ul style="text-align:left"> | ||
+ | <li>PCR amplification using Takara Ex Taq Polymerase testing different annealing temperatures.</li> | ||
+ | <li>Gel Migration on a 1% agarose gel.</li> | ||
+ | </ul> | ||
+ | |||
+ | <center><h3><em>PCR amplification of pSB1C3</em></h3></center></br> | ||
+ | <ul style="text-align:left"> | ||
+ | <li>PCR amplification using Takara Ex Taq Polymerase testing 8 annealing temperatures.</li> | ||
+ | </ul> | ||
+ | <table border="1" cellspacing="0" cellpadding="0" width="400"> | ||
+ | <tr> | ||
+ | <td width="133" valign="top"><p> </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> </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> | ||
+ | <br/> | ||
+ | |||
+ | <h3>Gel Migration on a 1% Agarose Gel</h3> | ||
+ | <p>Enzymatic digest of BBa_K808030</p> | ||
+ | <ul> | ||
+ | <li>Enzymatic digest by XbaI and Spe I of BBa_K808030</li> | ||
+ | <li>Gel migration.</li> | ||
+ | </ul> | ||
+ | <br /> | ||
+ | <center><h3><em>Yeast assembly</em></h3></center></br> | ||
+ | <p>The bacteria did not grow because the wrong antibiotic was chosen: BBa_J61047 was in pSB1A2 and not pSB1C3.</p> | ||
+ | <ul> | ||
+ | <li>Bacterial culture of BBa_J61047 on liquid LB + Amp 1X at 180 rpm, 37°C. The bacteria grew well.</li> | ||
+ | <li>Midi-prep has been done with the 50 ml of BBa_J61047 culture. (dilution 1/1000)</li> | ||
+ | </ul> | ||
+ | <table border="1" cellspacing="0" cellpadding="0" width="604"> | ||
+ | <tr> | ||
+ | <td width="121" valign="top"><p> </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(260/280nm)</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> | ||
+ | <br/> | ||
+ | <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’t work.</li> | ||
+ | <li>XbaI and SpeI digestion of BBa_J61047.</li> | ||
+ | </ul> | ||
+ | |||
+ | <ul style="text-align:left"> | ||
+ | <li>tube 1 « Miniprep XbaI/SpeI » : 5 µl 10X buffer cutsmart + 0.5 µL XbaI + 0.5 µl SpeI + 38 µl H<sub>2</sub>O +5 µl DNA, digestion at 37°C during 1 hour.</li> | ||
+ | <li>tube 2 « Midiprep XbaI/SpeI » : 5 µl 10X buffer cut smart + 0.5 µl XbaI + 0.5 µl SpeI + 38 µl H<sub>2</sub>O + 5 µl DNA, digestion at 37°C during 1 hour.</li> | ||
+ | <li>tube 3 « Miniprep NotI » : 5 µl 10X buffer cutsmart + 0.5 µl NotI + 38.5 µL H<sub>2</sub>O + 5 µL DNA, digestion at 37°C during 1 hour.</li> | ||
+ | <li>tube 4 « MiDiprep NotI » : 5 µl 10X buffer cutsmart + 0.5 µl NotI + 38.5 µL H<sub>2</sub>O + 5 µL DNA, digestion at 37°C during 1 hour.</li> | ||
+ | <li>tube 5 « Miniprep CTRL » as negative control : 5 µl DNA + 40 µl H<sub>2</sub>O + 5 µl 10X buffer cutsmart.</li> | ||
+ | <li>tube 6 « Midiprep CTRL » as negative control : 5 µl DNA + 40 µl H<sub>2</sub>O + 5 µl 10X buffer cutsmart.</li> | ||
+ | <li>MWT : « Quick-Load Purple 2-Log DNA Ladder (0.1-10.0 kb) NEB</li> | ||
+ | </ul> | ||
+ | <br/> | ||
+ | <br/> | ||
+ | |||
+ | <center><h3><em>PCR Amplification of the BBa_J61047 using Phusion Polymerase Master Mix.</em></h3></center></br> | ||
+ | <ul style="text-align:left"> | ||
+ | <li>Gel Migration on a 0.8% agarose gel</li> | ||
+ | <li>Culture of the plasmid pRS415 in SC_Leu at 30°C at 150 rpm for about 72 hours.</li> | ||
+ | <li>PCR Amplification using the Phusion Polymerase of pRS415.</li> | ||
+ | <li>Gel Extraction of the PCR products. </li> | ||
+ | </ul> | ||
+ | <br/> | ||
+ | <br/> | ||
+ | |||
+ | <center><h3><em>TPA solubility</em></h3></center></br> | ||
+ | <ul> | ||
+ | <li>Determination of the solubilization products of TPA.</li> | ||
+ | <li>Detection of 2-hydroxy-terephthalate using a TECAN spectrometer.</li> | ||
+ | <li>Precipitation of terephthalic acid in H<sub>2</sub>SO<sub>4</sub>.</li> | ||
+ | </ul> | ||
+ | |||
− | |||
− | + | <br> | |
+ | <br> | ||
− | |||
<a class="hautdepage" href="#"><p><span style="font-size: 60px; font-family: Courier;">^</span><br/><span style="font-size: 20px;">Page up</span></p></a></p> | <a class="hautdepage" href="#"><p><span style="font-size: 60px; font-family: Courier;">^</span><br/><span style="font-size: 20px;">Page up</span></p></a></p> | ||
</td> | </td> |
Latest revision as of 23:42, 3 October 2015
Week 1Week 2Week 3Week 4Week 5Week 6Week 7Week 8Week 9Week 10Week 11Week 12Week 13Week 14Week 15 |
Week 12 NB-Esterase Assay
pNP-Assay
Gibson AssemblyOD measurement of Cluster 4:
Gel plan:
DNA concentration assay
Gibson Assembly
Cluster 4A Gibson assembly
PCR amplification of Biobricks BBa_K808007, BBa_K808030, BBa_K936011, BBa_K936023, BBa_K1392932, BBa_K316003
The PCR worked for most of our Biobricks with the exception of the biobrick K808030. DNA concentration measurement
PCR amplification of pSB1C3
PCR amplification of pSB1C3
Cycles:
PCR amplification using Takara Ex TaqPCR amplification of Biobricks K808030Gel Plan:
PCR amplification of pSB1C3PCR amplification of pSB1C3
Gel Migration on a 1% Agarose GelEnzymatic digest of BBa_K808030 Yeast assemblyThe bacteria did not grow because the wrong antibiotic was chosen: BBa_J61047 was in pSB1A2 and not pSB1C3.
PCR Amplification of the BBa_J61047 using Phusion Polymerase Master Mix.TPA solubility^ |