Difference between revisions of "Team:Pasteur Paris/Week 12"
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− | + | ||
− | <h3 | + | <center><h3><em> NB-Esterase Assay </em></h3></center></br> |
− | < | + | <ul style="text-align:left"> |
− | < | + | <li>MiniPrep 808030 in the plasmid pDG011.</li> |
− | <h3 | + | </ul></br> |
+ | |||
+ | <center><h3><em>pNP-Assay</em></h3></center></br> | ||
<ul style="text-align:left"> | <ul style="text-align:left"> | ||
<li>Culture of DH5-α transformed with 808030 in pDG011</li> | <li>Culture of DH5-α transformed with 808030 in pDG011</li> | ||
Line 113: | Line 116: | ||
</ul> | </ul> | ||
<br/> | <br/> | ||
− | <h3 | + | <center><h3><em>Gibson Assembly</em></h3></center></br> |
<p style="text-align:left">OD measurement of Cluster 4:</p> | <p style="text-align:left">OD measurement of Cluster 4:</p> | ||
<table border="1" cellspacing="0" cellpadding="0" width="651"> | <table border="1" cellspacing="0" cellpadding="0" width="651"> | ||
Line 163: | Line 166: | ||
<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 | + | <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> | ||
Line 223: | Line 226: | ||
<br/> | <br/> | ||
− | < | + | <center><h3><em>DNA concentration assay</em></h3></center></br> |
<table border="1" cellspacing="0" cellpadding="0" width="678"> | <table border="1" cellspacing="0" cellpadding="0" width="678"> | ||
Line 284: | Line 287: | ||
− | <h3 | + | <center><h3><em>Gibson Assembly</em></h3></center></br> |
<ul style="text-align:left"> | <ul style="text-align:left"> | ||
<li>Enzymatic digest of K936011, K936023 and K1392932</li> | <li>Enzymatic digest of K936011, K936023 and K1392932</li> | ||
Line 293: | Line 296: | ||
<br /> | <br /> | ||
− | <h3 | + | <center><h3><em>Cluster 4A Gibson assembly</em></h3></center></br> |
<ul style="text-align:left"> | <ul style="text-align:left"> | ||
<li>Gibson Assembly</li> | <li>Gibson Assembly</li> | ||
<li>PCR amplification of cluster 4A Assembly.</li> | <li>PCR amplification of cluster 4A Assembly.</li> | ||
<li>Gel migration on 0.8% agarose gel.</li> | <li>Gel migration on 0.8% agarose gel.</li> | ||
+ | <li>Gel plan</em></li> | ||
</ul> | </ul> | ||
− | <br | + | </br> |
− | + | ||
<table border="1" cellspacing="0" cellpadding="0" width="642"> | <table border="1" cellspacing="0" cellpadding="0" width="642"> | ||
Line 321: | Line 324: | ||
</table> | </table> | ||
<br/> | <br/> | ||
− | <h3 | + | |
+ | <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"> | <ul style="text-align:left"> | ||
<li>PCR amplification using Phusion polymerase</li> | <li>PCR amplification using Phusion polymerase</li> | ||
Line 334: | Line 338: | ||
<tr> | <tr> | ||
<td width="64" valign="top"><p>1/25 dilution</p></td> | <td width="64" valign="top"><p>1/25 dilution</p></td> | ||
− | <td width="77" valign="top"><p>DNA concentration (ng/ | + | <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(230nm)</p></td> | ||
<td width="77" valign="top"><p>OD(260nm)</p></td> | <td width="77" valign="top"><p>OD(260nm)</p></td> | ||
Line 374: | Line 378: | ||
<tr> | <tr> | ||
<td width="64" valign="top"><p align="right">936023</p></td> | <td width="64" valign="top"><p align="right">936023</p></td> | ||
− | <td width="77" valign="top"><p align="right">5 | + | <td width="77" valign="top"><p align="right">5.2</p></td> |
− | <td width="77" valign="top"><p align="right">0 | + | <td width="77" valign="top"><p align="right">0.097</p></td> |
− | <td width="77" valign="top"><p align="right">0 | + | <td width="77" valign="top"><p align="right">0.105</p></td> |
− | <td width="77" valign="top"><p align="right">0 | + | <td width="77" valign="top"><p align="right">0.062</p></td> |
− | <td width="77" valign="top"><p align="right">0 | + | <td width="77" valign="top"><p align="right">0.003</p></td> |
− | <td width="77" valign="top"><p align="right">1 | + | <td width="77" valign="top"><p align="right">1.7</p></td> |
− | <td width="77" valign="top"><p align="right">1 | + | <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="64" valign="top"><p align="right">1392932</p></td> | ||
− | <td width="77" valign="top"><p align="right">12 | + | <td width="77" valign="top"><p align="right">12.6</p></td> |
− | <td width="77" valign="top"><p align="right">0 | + | <td width="77" valign="top"><p align="right">0.493</p></td> |
− | <td width="77" valign="top"><p align="right">0 | + | <td width="77" valign="top"><p align="right">0.251</p></td> |
− | <td width="77" valign="top"><p align="right">0 | + | <td width="77" valign="top"><p align="right">0.259</p></td> |
− | <td width="77" valign="top"><p align="right">0 | + | <td width="77" valign="top"><p align="right">0.035</p></td> |
− | <td width="77" valign="top"><p align="right">0 | + | <td width="77" valign="top"><p align="right">0.97</p></td> |
− | <td width="77" valign="top"><p align="right">0 | + | <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="64" valign="top"><p align="right">316003</p></td> | ||
− | <td width="77" valign="top"><p align="right">5 | + | <td width="77" valign="top"><p align="right">5.5</p></td> |
− | <td width="77" valign="top"><p align="right">0 | + | <td width="77" valign="top"><p align="right">0.1</p></td> |
− | <td width="77" valign="top"><p align="right">0 | + | <td width="77" valign="top"><p align="right">0.11</p></td> |
− | <td width="77" valign="top"><p align="right">0 | + | <td width="77" valign="top"><p align="right">0.062</p></td> |
− | <td width="77" valign="top"><p align="right">0 | + | <td width="77" valign="top"><p align="right">0.0</p></td> |
− | <td width="77" valign="top"><p align="right">1 | + | <td width="77" valign="top"><p align="right">1.76</p></td> |
− | <td width="77" valign="top"><p align="right">1 | + | <td width="77" valign="top"><p align="right">1.10</p></td> |
</tr> | </tr> | ||
</table> | </table> | ||
<br /> | <br /> | ||
− | + | <center><h3><em>PCR amplification of pSB1C3</em></h3></center></br> | |
<ul style="text-align:left"> | <ul style="text-align:left"> | ||
<li>PCR amplification using Phusion Polymerase.</li> | <li>PCR amplification using Phusion Polymerase.</li> | ||
Line 432: | Line 436: | ||
</table> | </table> | ||
<br/> | <br/> | ||
− | <h3 | + | |
+ | <center><h3><em>PCR amplification of pSB1C3</em></h3></center></br> | ||
<ul style="text-align:left"> | <ul style="text-align:left"> | ||
<li>PCR amplification using Takara Ex Taq Polymerase testing different annealing temperatures.</li> | <li>PCR amplification using Takara Ex Taq Polymerase testing different annealing temperatures.</li> | ||
Line 446: | Line 451: | ||
<tr> | <tr> | ||
<td width="133" valign="top"><p>DNAse, RNAse free water</p></td> | <td width="133" valign="top"><p>DNAse, RNAse free water</p></td> | ||
− | <td width="77" valign="top"><p align="right">41 | + | <td width="77" valign="top"><p align="right">41.75</p></td> |
− | <td width="71" valign="top"><p align="right">40 | + | <td width="71" valign="top"><p align="right">40.75</p></td> |
− | <td width="73" valign="top"><p align="right">40 | + | <td width="73" valign="top"><p align="right">40.75</p></td> |
− | <td width="45" valign="top"><p align="right">39 | + | <td width="45" valign="top"><p align="right">39.75</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
Line 488: | Line 493: | ||
<tr> | <tr> | ||
<td width="133" valign="top"><p>Ex Taq Polymerase</p></td> | <td width="133" valign="top"><p>Ex Taq Polymerase</p></td> | ||
− | <td width="77" valign="top"><p align="right">0 | + | <td width="77" valign="top"><p align="right">0.25</p></td> |
− | <td width="71" valign="top"><p align="right">0 | + | <td width="71" valign="top"><p align="right">0.25</p></td> |
− | <td width="73" valign="top"><p align="right">0 | + | <td width="73" valign="top"><p align="right">0.25</p></td> |
− | <td width="45" valign="top"><p align="right">0 | + | <td width="45" valign="top"><p align="right">0.25</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
Line 509: | Line 514: | ||
<li>annealing for 30s:</li> | <li>annealing for 30s:</li> | ||
<li>60°C: Tube 1-4</li> | <li>60°C: Tube 1-4</li> | ||
− | <li>61 | + | <li>61.1°C: Tube 5-8</li> |
<li>63°C: Tube 9-12</li> | <li>63°C: Tube 9-12</li> | ||
− | <li>65 | + | <li>65.6°C: Tube 13-16</li> |
− | <li>69 | + | <li>69.2°C: Tube 17-20</li> |
− | <li>72 | + | <li>72.1°C: Tube 21-24</li> |
− | <li>73 | + | <li>73.9°C: Tube 25-28</li> |
<li>75°C: Tube 29-32</li> | <li>75°C: Tube 29-32</li> | ||
<li>72°C for 80s</li> | <li>72°C for 80s</li> | ||
Line 520: | Line 525: | ||
<br/> | <br/> | ||
<br/> | <br/> | ||
− | <h3 | + | |
+ | <center><h3><em>PCR amplification using Takara Ex Taq</em></h3></center></br> | ||
<ul style="text-align:left"> | <ul style="text-align:left"> | ||
− | <li>Gel Migration on a 1% | + | <li>Gel Migration on a 1% agarose gel.</li> |
</ul> | </ul> | ||
− | <h3 | + | <center><h3><em>PCR amplification of Biobricks K808030</em></h3></center></br> |
<ul style="text-align:left"> | <ul style="text-align:left"> | ||
<li>PCR Amplification using Takara Ex Taq Polymerase.</li> | <li>PCR Amplification using Takara Ex Taq Polymerase.</li> | ||
Line 553: | Line 559: | ||
<br /> | <br /> | ||
<br /> | <br /> | ||
− | <h3 | + | <center><h3><em>PCR amplification of pSB1C3</em></h3></center></br> |
<ul style="text-align:left"> | <ul style="text-align:left"> | ||
<li>PCR amplification using Takara Ex Taq Polymerase testing different annealing temperatures.</li> | <li>PCR amplification using Takara Ex Taq Polymerase testing different annealing temperatures.</li> | ||
− | <li>Gel Migration on a 1% | + | <li>Gel Migration on a 1% agarose gel.</li> |
</ul> | </ul> | ||
− | <h3 | + | <center><h3><em>PCR amplification of pSB1C3</em></h3></center></br> |
<ul style="text-align:left"> | <ul style="text-align:left"> | ||
<li>PCR amplification using Takara Ex Taq Polymerase testing 8 annealing temperatures.</li> | <li>PCR amplification using Takara Ex Taq Polymerase testing 8 annealing temperatures.</li> | ||
Line 573: | Line 579: | ||
<tr> | <tr> | ||
<td width="133" valign="top"><p>DNAse, RNAse free water</p></td> | <td width="133" valign="top"><p>DNAse, RNAse free water</p></td> | ||
− | <td width="77" valign="top"><p align="right">41 | + | <td width="77" valign="top"><p align="right">41.75</p></td> |
− | <td width="71" valign="top"><p align="right">40 | + | <td width="71" valign="top"><p align="right">40.75</p></td> |
− | <td width="73" valign="top"><p align="right">40 | + | <td width="73" valign="top"><p align="right">40.75</p></td> |
− | <td width="45" valign="top"><p align="right">39 | + | <td width="45" valign="top"><p align="right">39.75</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
Line 615: | Line 621: | ||
<tr> | <tr> | ||
<td width="133" valign="top"><p>Ex Taq Polymerase</p></td> | <td width="133" valign="top"><p>Ex Taq Polymerase</p></td> | ||
− | <td width="77" valign="top"><p align="right">0 | + | <td width="77" valign="top"><p align="right">0.25</p></td> |
− | <td width="71" valign="top"><p align="right">0 | + | <td width="71" valign="top"><p align="right">0.25</p></td> |
− | <td width="73" valign="top"><p align="right">0 | + | <td width="73" valign="top"><p align="right">0.25</p></td> |
− | <td width="45" valign="top"><p align="right">0 | + | <td width="45" valign="top"><p align="right">0.25</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
Line 637: | Line 643: | ||
</ul> | </ul> | ||
<br /> | <br /> | ||
− | <h3>Yeast | + | <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> | <p>The bacteria did not grow because the wrong antibiotic was chosen: BBa_J61047 was in pSB1A2 and not pSB1C3.</p> | ||
<ul> | <ul> | ||
<li>Bacterial culture of BBa_J61047 on liquid LB + Amp 1X at 180 rpm, 37°C. The bacteria grew well.</li> | <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 | + | <li>Midi-prep has been done with the 50 ml of BBa_J61047 culture. (dilution 1/1000)</li> |
</ul> | </ul> | ||
<table border="1" cellspacing="0" cellpadding="0" width="604"> | <table border="1" cellspacing="0" cellpadding="0" width="604"> | ||
Line 653: | Line 659: | ||
<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 | + | <td width="121" valign="top"><p align="right">0.5</p></td> |
− | <td width="121" valign="top"><p align="right">0 | + | <td width="121" valign="top"><p align="right">0.011</p></td> |
− | <td width="121" valign="top"><p align="right">0 | + | <td width="121" valign="top"><p align="right">0.004</p></td> |
− | <td width="121" valign="top"><p align="right">2 | + | <td width="121" valign="top"><p align="right">2.57</p></td> |
</tr> | </tr> | ||
</table> | </table> | ||
<br/> | <br/> | ||
<ul style="text-align:left"> | <ul style="text-align:left"> | ||
− | <li> Preparation of YPD specific media on specific media (1% yeast extract, 2% | + | <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>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 | + | <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> | <li>XbaI and SpeI digestion of BBa_J61047.</li> | ||
</ul> | </ul> | ||
<ul style="text-align:left"> | <ul style="text-align:left"> | ||
− | <li>tube 1 « Miniprep XbaI/SpeI » : | + | <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 » : | + | <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 » : | + | <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 » : | + | <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 : | + | <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 : | + | <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> | <li>MWT : « Quick-Load Purple 2-Log DNA Ladder (0.1-10.0 kb) NEB</li> | ||
</ul> | </ul> | ||
Line 680: | Line 686: | ||
<br/> | <br/> | ||
− | <h3 | + | <center><h3><em>PCR Amplification of the BBa_J61047 using Phusion Polymerase Master Mix.</em></h3></center></br> |
<ul style="text-align:left"> | <ul style="text-align:left"> | ||
<li>Gel Migration on a 0.8% agarose gel</li> | <li>Gel Migration on a 0.8% agarose gel</li> | ||
− | <li>Culture of the plasmid pRS415 in SC_Leu at 30°C at | + | <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>PCR Amplification using the Phusion Polymerase of pRS415.</li> | ||
<li>Gel Extraction of the PCR products. </li> | <li>Gel Extraction of the PCR products. </li> | ||
Line 689: | Line 695: | ||
<br/> | <br/> | ||
<br/> | <br/> | ||
− | <h3 | + | |
+ | <center><h3><em>TPA solubility</em></h3></center></br> | ||
<ul> | <ul> | ||
<li>Determination of the solubilization products of TPA.</li> | <li>Determination of the solubilization products of TPA.</li> | ||
<li>Detection of 2-hydroxy-terephthalate using a TECAN spectrometer.</li> | <li>Detection of 2-hydroxy-terephthalate using a TECAN spectrometer.</li> | ||
− | <li> | + | <li>Precipitation of terephthalic acid in H<sub>2</sub>SO<sub>4</sub>.</li> |
</ul> | </ul> | ||
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^ |