Team:Exeter/lab book/3gip/wk10

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         </p>
         </p>
         <a href="https://2012.igem.org/Team:Exeter/lab_book/3gip/wk11"; style="color:#1d1d1b">17th - 21st September</a>
         <a href="https://2012.igem.org/Team:Exeter/lab_book/3gip/wk11"; style="color:#1d1d1b">17th - 21st September</a>
 +
        <p>
 +
        -
 +
        </p>
 +
        <a href="https://2012.igem.org/Team:Exeter/Results/inducible"; style="color:#e30614" target="_blank"><font size="3"><b>Results</b></font></a>
 +
</font>
       </font>
       </font>
     </div>
     </div>
     <!--End Project Division Week Hyperlinks-->
     <!--End Project Division Week Hyperlinks-->
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     </td>
     </td>
    
    
   <td width="850" height="250">
   <td width="850" height="250">
   <!------------INSERT WEEKLY IMAGE HERE------------>
   <!------------INSERT WEEKLY IMAGE HERE------------>
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     <img src="" alt="" title="" width="850" height="250">
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     <img src="https://static.igem.org/mediawiki/2012/1/1c/Exe2012Freddielookingpensive.JPG" alt="" title="" width="850" height="250">
   </td>
   </td>
   </tr>
   </tr>
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   <td valign="top" width="850">
   <td valign="top" width="850">
     <div style="text-align:justify">
     <div style="text-align:justify">
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     <font face="DokChampa" color="#1d1d1b" size="2">
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     <font face="Verdana" color="#1d1d1b" size="2">
      
      
-
       <font face="DokChampa" color="#57b947" size="4">
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       <font face="Verdana" color="#57b947" size="4">
       <p><b><u>The 3-Gene Inducible Plasmid: 10th - 14th September 2012</u></b></p>
       <p><b><u>The 3-Gene Inducible Plasmid: 10th - 14th September 2012</u></b></p>
       </font>
       </font>
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     <p><b><u>Monday 10th September</u></b></p>
     <p><b><u>Monday 10th September</u></b></p>
<p><i><b>Morning</b></i></p>
<p><i><b>Morning</b></i></p>
-
<p>•<u>3A assembly</u> </p>  
+
<p>•<a href="https://2012.igem.org/Team:Exeter/lab_book/proto/4" style="color:#57b947"><u>3A Assembly</u></a></p>  
-
<p>BBa_K206000_BBaB0034 + ompA_BBa_K322921_BBa_B0014 in pSB1C3 (1) </p>
+
<p>BBa_K206000_BBaB0034 + <i>ompA</i> BBa_K322921_BBa_B0014 in pSB1C3 (1) </p>
-
<p>BBa_K206000_BBaB0034 + ompA_BBa_K322921_BBa_B0014 in pSB1C3 (2) </p>
+
<p>BBa_K206000_BBaB0034 + <i>ompA</i> BBa_K322921_BBa_B0014 in pSB1C3 (2) </p>
-
<p>BBa_J13002 + ompA_BBa_K322921_BBa_B0014 in pSB1C3 (1) </p>
+
<p>BBa_J13002 + <i>ompA</i> BBa_K322921_BBa_B0014 in pSB1C3 (1) </p>
-
<p>BBa_J13002 + ompA_BBa_K322921_BBa_B0014 in pSB1C3 (2) </p>
+
<p>BBa_J13002 + <i>ompA</i> BBa_K322921_BBa_B0014 in pSB1C3 (2) </p>
-
<p>BBa_J23119 + WbnJ_BBa_B0014 in pSB1C3</p>
+
<p>BBa_J23119 + <i>wbnJ</i> BBa_B0014 in pSB1C3</p>
-
<p>BBa_B0034 + WfcA_ BBaB0014 in pSB1C3</p>
+
<p>BBa_B0034 + <i>wfcA</i> BBaB0014 in pSB1C3</p>
-
<p>BBa_B0034 + WfcA_BBaB0014 in pSB1K3</p>
+
<p>BBa_B0034 + <i>wfcA</i> BBaB0014 in pSB1K3</p>
-
<p>BBaK094120_BBa_B0034 + WfcA_BBaB0014 in pSB1C3</p>
+
<p>BBaK094120_BBa_B0034 + <i>wfcA</i> BBaB0014 in pSB1C3</p>
-
<p>Performed with the NEB protocol with some adaptions again. The volume of digestion was changed from 50μl to 20μl by adding less water so as to increase the DNA concentration. The enzymes and buffer concentrations were adapted as below. This was not a Linearized Plasmid Backbone so for the plasmid digestion we used the NEB Biolabs protocol adapted to the amounts shown below. </p>
+
<p>Performed with the NEB protocol with some adaptations again. The volume of digestion was changed from 50μl to 20μl by adding less water so as to increase the DNA concentration. The enzymes and buffer concentrations were adapted as below. This was not a linearized plasmid backbone so for the plasmid digestion we used the NEB Biolabs protocol adapted to the amounts shown below. </p><br>
-
<p>
+
-
</p>
+
-
.
+
<p><b><u>Tuesday 11th September</u></b></p>
<p><b><u>Tuesday 11th September</u></b></p>
<p><i><b>Morning</b></i></p>
<p><i><b>Morning</b></i></p>
-
<p>•<u>Converting Biobricks into pSB1C3</u></p>
+
<p>•<a href="https://2012.igem.org/Team:Exeter/lab_book/proto/4" style="color:#57b947"><u>Transferring BioBricks</u></a> <u>into pSB1C3</u></p>
-
<p>WbnJ into pSB1C3</p>
+
<p><i>wbnJ</i> into pSB1C3</p>
-
<p>WbnK into pSB1C3</p>
+
<p><i>wbnK</i> into pSB1C3</p>
-
<p>WfcA into pSB1C3</p>
+
<p><i>wfcA</i> into pSB1C3</p>
-
<p>WbbC(1) into pSB1C3</p>
+
<p><i>wbbC</i>(1) into pSB1C3</p>
-
<p>WbbC(2) into pSB1C3</p>
+
<p><i>wbbC</i>(2) into pSB1C3</p>
-
<p>WbbC(3) into pSB1C3</p>
+
<p><i>wbbC</i>(3) into pSB1C3</p>
-
<p>WclY into pSB1C3</p>
+
<p><i>wclY</i> into pSB1C3</p>
-
<p>WbiP into pSB1C3</p>
+
<p><i>wbiP</i> into pSB1C3</p>
-
<p>WbiP into pSB1C3</p>
+
<p><i>wbiP</i> into pSB1C3</p>
-
<p>The genes and the destination plasmid were cut using EcoR1 and PstI. The digest was incubated for an hour and a half and then deactivated by heating at 80C for 20 minutes. The ligation was performed as per the 3A assembly above but 4ul of plasmid DNA was used to 2ul of destination plasmid. </p>
+
<p>The genes and the destination plasmid were cut using EcoR1 and PstI. The digest was incubated for 90 minutes and then deactivated by heating at 80°C for 20 minutes. The ligation was performed as per the 3A assembly above but 4µl of plasmid DNA was used to 2µl of destination plasmid. </p><br>
-
<p>
+
 
-
</p>
+
<p><i><b>Afternoon</b></i></p>
<p><i><b>Afternoon</b></i></p>
-
<p>•<u>Transformation</u></p>
+
<p>•<a href="https://2012.igem.org/Team:Exeter/lab_book/proto/1" style="color:#57b947"><u>Transformation</u></a></p>
-
<p>The ligations above and the ligations from the 12/09/2012 were transformed using the Competent Cell protocol and using the Competent Cells made in the lab. </p>
+
<p>The ligations above and the ligations from the 12/09/2012 were transformed using the Competent Cell protocol using our competent cells made in the lab. </p><br>
<p><b><u>Wednesday 12th September</u></b></p>
<p><b><u>Wednesday 12th September</u></b></p>
<p><i><b>Morning</b></i></p>
<p><i><b>Morning</b></i></p>
-
<p>WbbC(1) into pSB1C3, WbbC(2) into pSB1C3, WbbC(3) into pSB1C3 and WbiP into pSB1C3 did not form colonies on the plates. This was repeated. </p>
+
<p><i>wbbC</i>(1) into pSB1C3, <i>wbbC</i>(2) into pSB1C3, <i>wbbC</i>(3) into pSB1C3 and <i>wbiP</i> into pSB1C3 did not form colonies on the plates. They were repeated. </p><br>
-
<p>•<u>Converting Biobricks into pSB1C3</u></p>
+
-
<p>ompA into pSB1C3</p>
+
-
<p>WbbC(1) into pSB1C3</p>
+
-
<p>WbbC(2) into pSB1C3</p>
+
-
<p>WbbC(3) into pSB1C3</p>
+
-
<p>WbiP into pSB1C3</p>
+
-
<p>The genes and the destination plasmid were cut using EcoR1 and PstI. The digest was incubated for an hour and a half and then deactivated by heating at 80C for 20 minutes. The ligation was performed as per the 3A assembly above but 4μl of plasmid DNA was used to 2μl of destination plasmid. </p>
+
-
<p>
+
-
</p>
+
-
<p>•<u>3A assembly</u></p> 
+
-
<p>ompA + Cyc_BBa_B0014</p>
+
-
<p>Performed with the NEB protocol with some adaptions again. The volume of digestion was changed from 50μl to 20μl by adding less water so as to increase the DNA concentration. The enzymes and buffer concentrations were adapted as below. This was not a Linearized Plasmid Backbone so for the plasmid digestion we used the NEB Biolabs protocol adapted to the amounts shown below. </p>
+
-
<p>
+
-
</p>
+
-
<p>•<u>Two step Phusion PCR using a 6 X 10 serial dilution</u></p>
+
<p>•<a href="https://2012.igem.org/Team:Exeter/lab_book/proto/4" style="color:#57b947"><u>Transferring BioBricks</u></a> <u>into pSB1C3</u></p>
 +
<p><i>ompA</i> into pSB1C3</p>
 +
<p><i>wbbC</i>(1) into pSB1C3</p>
 +
<p><i>wbbC</i>(2) into pSB1C3</p>
 +
<p><i>wbbC</i>(3) into pSB1C3</p>
 +
<p><i>wbiP</i> into pSB1C3</p>
 +
<p>The genes and the destination plasmid were cut using EcoR1 and PstI. The digest was incubated for 90 minutes and then deactivated by heating at 80°C for 20 minutes. The ligation was performed as per the 3A assembly above but 4μl of plasmid DNA was used to 2μl of destination plasmid. </p><br>
-
<p>The reaction was set up using the Biolabs Phusion PCR protocol. </p>
+
<p>•<a href="https://2012.igem.org/Team:Exeter/lab_book/proto/4" style="color:#57b947"><u>3A Assembly</u></a></p> 
-
<p>
+
<p><i>ompA</i> + <i>amyA</i> BBa_B0014</p>
 +
<p>Performed with the NEB protocol with some adaptations again. The volume of digestion was changed from 50μl to 20μl by adding less water so as to increase the DNA concentration. The enzymes and buffer concentrations were adapted as below. This was not a linearized plasmid backbone so for the plasmid digestion we used the NEB Biolabs protocol adapted to the amounts shown below. </p><br>
-
</p>
+
<p>•<a href="https://2012.igem.org/Team:Exeter/lab_book/proto/7" style="color:#57b947"><u>Two Step Phusion PCR</u></a> <u>using a 6 X 10 serial dilution</u></p>
 +
 
 +
<p>The reaction was set up using the Biolabs Phusion PCR protocol. </p><br>
<p>Two reactions were set up: </p>
<p>Two reactions were set up: </p>
-
<p>Reaction 1: Pbad Large and WbnJ using the primers supplied. See, Operon Construction: 9th - 13th July 2012</p>
+
<p>Reaction 1: Pbad Large and <i>wbnJ</i> using the primers supplied. See <a href="https://2012.igem.org/Team:Exeter/lab_book/gibs/wk1" style="color:#57b947" target="_blank"><u><b>Operon Construction:</b></u></a> 9th - 13th July 2012</p>
-
<p>Reaction 2: Reaction 1: Pbad Large and WbnJ using the primers supplied and varying the magnesium concentration. See, Operon Construction: 9th - 13th July 2012</p>
+
<p>Reaction 2: Reaction 1: Pbad Large and <i>wbnJ</i> using the primers supplied and varying the magnesium concentration. See <a href="https://2012.igem.org/Team:Exeter/lab_book/gibs/wk1" style="color:#57b947" target="_blank"><u><b>Operon Construction:</b></u></a> 9th - 13th July 2012</p>
-
<p>PCR mix and this was loaded onto the gel and run for 20 minutes.
+
<p>After PCR, PCR mix was dyed, loaded onto the gel and run for 20 minutes.
-
No bands were shown on the gel so the PCR did not work. </p>
+
No bands were shown on the gel so the PCR did not work. </p><br>
<p><i><b>Afternoon</b></i></p>
<p><i><b>Afternoon</b></i></p>
-
<p>•<u>Adding cultures</u></p>   
+
<p>•<a href="https://2012.igem.org/Team:Exeter/lab_book/proto/2" style="color:#57b947"><u>Transferring colonies to liquid medium</u></a></p>   
<p>Cells transformed with below were added into liquid medium and incubated overnight. </p>
<p>Cells transformed with below were added into liquid medium and incubated overnight. </p>
<p>BBa_E0240</p>
<p>BBa_E0240</p>
-
<p>BBa_K206000_BBaB0034 + ompA_BBa_K322921_BBa_B0014 in pSB1C3 (1) </p>
+
<p>BBa_K206000_BBaB0034 + <i>ompA</i> BBa_K322921_BBa_B0014 in pSB1C3 (1) </p>
-
<p>BBa_K206000_BBaB0034 + ompA_BBa_K322921_BBa_B0014 in pSB1C3 (2) </p>
+
<p>BBa_K206000_BBaB0034 + <i>ompA</i> BBa_K322921_BBa_B0014 in pSB1C3 (2) </p>
-
<p>BBa_J13002 + ompA_BBa_K322921_BBa_B0014 in pSB1C3 (1) </p>
+
<p>BBa_J13002 + <i>ompA</i> BBa_K322921_BBa_B0014 in pSB1C3 (1) </p>
-
<p>BBa_J13002 + ompA_BBa_K322921_BBa_B0014 in pSB1C3 (2) </p>
+
<p>BBa_J13002 + <i>ompA</i> BBa_K322921_BBa_B0014 in pSB1C3 (2) </p>
-
<p>BBa_J23119 + WbnJ_BBa_B0014 in pSB1C3</p>
+
<p>BBa_J23119 + <i>wbnJ</i> BBa_B0014 in pSB1C3</p>
-
<p>BBa_B0034 + WfcA_BBaB0014 in pSB1C3</p>
+
<p>BBa_B0034 + <i>wfcA</i> BBaB0014 in pSB1C3</p>
-
<p>BBaK094120_BBa_B0034 + WfcA_BBaB0014 in pSB1C3</p>
+
<p>BBaK094120_BBa_B0034 + <i>wfcA</i> BBaB0014 in pSB1C3</p>
-
<p>WbnJ into pSB1C3</p>
+
<p><i>wbnJ</i> into pSB1C3</p>
-
<p>WbnK into pSB1C3</p>
+
<p><i>wbnK</i> into pSB1C3</p>
-
<p>WfcA into pSB1C3</p>
+
<p><i>wfcA</i> into pSB1C3</p>
-
<p>WclY into pSB1C3</p>
+
<p><i>wclY</i> into pSB1C3</p>
-
<p>WbiP into pSB1C3</p>
+
<p><i>wbiP</i> into pSB1C3</p>
-
<p>Protocol was followed using chloramphenicol for the BioBrick part as the selection antibiotic. </p>
+
<p>Protocol was followed using chloramphenicol for the BioBrick part as the selection antibiotic. </p><br>
<p><b><u>Thursday 13th. September</u></b></p>
<p><b><u>Thursday 13th. September</u></b></p>
<p><i><b>Morning</b></i></p>
<p><i><b>Morning</b></i></p>
-
<p>ompA + Cyc_BBa_B0014 showed no colonies on the overnight plate. </p>
+
<p><i>ompA</i> + <i>amyA</i> BBa_B0014 showed no colonies on the overnight plate. </p>
-
<p>•<u>Mini-Prepping</u></p>   
+
<p>•<a href="http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_k0502.pdf" style="color:#57B947" target="_blank"><u>Miniprepping</u></a></p>   
<p>BBa_E0240</p>
<p>BBa_E0240</p>
-
<p>BBa_K206000_BBaB0034 + ompA_BBa_K322921_BBa_B0014 in pSB1C3 (1) </p>
+
<p>BBa_K206000_BBaB0034 + <i>ompA</i> BBa_K322921_BBa_B0014 in pSB1C3 (1) </p>
-
<p>BBa_K206000_BBaB0034 + ompA_BBa_K322921_BBa_B0014 in pSB1C3 (2) </p>
+
<p>BBa_K206000_BBaB0034 + <i>ompA</i> BBa_K322921_BBa_B0014 in pSB1C3 (2) </p>
-
<p>BBa_J13002 + ompA_BBa_K322921_BBa_B0014 in pSB1C3 (1) </p>
+
<p>BBa_J13002 + <i>ompA</i> BBa_K322921_BBa_B0014 in pSB1C3 (1) </p>
-
<p>BBa_J13002 + ompA_BBa_K322921_BBa_B0014 in pSB1C3 (2) </p>
+
<p>BBa_J13002 + <i>ompA</i> BBa_K322921_BBa_B0014 in pSB1C3 (2) </p>
-
<p>BBa_J23119 + WbnJ_BBa_B0014 in pSB1C3</p>
+
<p>BBa_J23119 + <i>wbnJ</i> BBa_B0014 in pSB1C3</p>
-
<p>BBa_B0034 + WfcA_BBaB0014 in pSB1C3</p>
+
<p>BBa_B0034 + <i>wfcA</i> BBaB0014 in pSB1C3</p>
-
<p>BBaK094120_BBa_B0034 + WfcA_BBaB0014 in pSB1C3</p>
+
<p>BBaK094120_BBa_B0034 + <i>WfcA</i> BBaB0014 in pSB1C3</p>
-
<p>WbnJ into pSB1C3</p>
+
<p><i>wbnJ</i> into pSB1C3</p>
-
<p>WbnK into pSB1C3</p>
+
<p><i>wbnK</i> into pSB1C3</p>
-
<p>WfcA into pSB1C3</p>
+
<p><i>wfcA</i> into pSB1C3</p>
-
<p>WclY into pSB1C3</p>
+
<p><i>wclY</i> into pSB1C3</p>
-
<p>WbiP into pSB1C3</p>
+
<p><i>wbiP</i> into pSB1C3</p>
-
<p>All minipreps were nanodropped showing very good concentrations</p>
+
<p>All mini-preps were nanodropped showing very good concentrations.</p><br>
 +
 
<p><i><b>Afternoon</b></i></p>
<p><i><b>Afternoon</b></i></p>
-
<p>•<u>Gel Digest of DNA from 21/08/2012</u></p>
+
<p>•<u>Gel</u> <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/4" style="color:#57b947"><u>Digest</u></a> <u>of DNA from 21/08/2012</u></p>
<p>A digestion using EcoR1 and PstI was run on all of the miniprepped DNA from today. </p>  
<p>A digestion using EcoR1 and PstI was run on all of the miniprepped DNA from today. </p>  
-
<p>For the digestion we used Fermantas enzymes instead. The protocol remained the same but we used Ferantas Buffer H instead of the NEB Buffer 2. </p>
+
<p>For the digestion we used Fermentas enzymes instead. The protocol remained the same but Fermentas Buffer H was used instead of the NEB Buffer 2. </p><br>
 +
 
<p><b><u>Friday 14th September</u></b></p>
<p><b><u>Friday 14th September</u></b></p>
-
<p>Today a digestion ligation was set up to put our remaining Biobrisks into pSB1C3. </p>
+
<p>Today a <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/4" style="color:#57b947"><u>digestion/ligation</u></a> was set up to put our remaining BioBricks into pSB1C3. </p>
-
<p>WbnJ</p>
+
<p><i>wbnJ</i></p>
<p>BBa_K094120_BBaB0034 in pSB1C3</p>
<p>BBa_K094120_BBaB0034 in pSB1C3</p>
-
<p>WclY in pSB1C3</p>
+
<p><i>wclY</i> in pSB1C3</p>
-
<p>Wbbc in pSB1C3</p>
+
<p><i>wbbc</i> in pSB1C3</p>
-
<p>WfcA in pSB1C3</p>
+
<p><i>wfcA</i> in pSB1C3</p>
-
<p>BBaB0034_WbiP in pSB1C3</p>
+
<p>BBaB0034_<i>wbiP</i> in pSB1C3</p>
-
<p>BBaB0034_WbnK in pSB1C3</p>
+
<p>BBaB0034_<i>wbnK</i> in pSB1C3</p>
-
<p>BBaB0034_WclY in pSB1C3</p>
+
<p>BBaB0034_<i>wclY</i> in pSB1C3</p>
<p>BBa_K206000_BBa_B0034 in pSB1C3</p>
<p>BBa_K206000_BBa_B0034 in pSB1C3</p>
<p>BBa_ K206001_BBa_B0034 in pSB1C3</p>
<p>BBa_ K206001_BBa_B0034 in pSB1C3</p>
<p>BBa_I0500_BBa_B0034 in pSB1C3</p>
<p>BBa_I0500_BBa_B0034 in pSB1C3</p>
<p>BBa_J23119_ BBa_B0034 in pSB1C3</p>
<p>BBa_J23119_ BBa_B0034 in pSB1C3</p>
-
<p>ompA in pSB1C3</p>
+
<p><i>ompA</i> in pSB1C3</p>
-
<p>ompA_K322921_BBa_B0014 in pSB1C3</p>
+
<p><i>ompA</i> K322921_BBa_B0014 in pSB1C3</p>
-
<p>BBa_K094120_BBa_B0034_WclY_BBa_B0014 in pSB1C3</p>
+
<p>BBa_K094120_BBa_B0034_<i>wclY</i> BBa_B0014 in pSB1C3</p>
-
<p>BBa_J13002 _BBa_B0034_WclY_BBa_B0014 in pSB1C3</p>
+
<p>BBa_J13002 _BBa_B0034_<i>wclY</i> BBa_B0014 in pSB1C3</p>
-
<p>BBa_J13002_Cyc_BBa_B0014 in pSB1C3</p>
+
<p>BBa_J13002 <i>amyA</i> BBa_B0014 in pSB1C3</p>
-
<p>BBa_J13002_Has_BBa_B0014 in pSB1C3</p>
+
<p>BBa_J13002 <i>hasA</i> BBa_B0014 in pSB1C3</p>
-
<p>The genes and the destination plasmid were cut using EcoR1 and PstI. The digest was incubated for an hour and a half and then deactivated by heating at 80C for 20 minutes. The ligation was performed as per the 3A assembly above but 4μl of plasmid DNA was used to 2μl of destination plasmid. </p>
+
<p>The genes and the destination plasmid were cut using EcoR1 and PstI. The digest was incubated for 90 minutes and then deactivated by heating at 80°C for 20 minutes. The ligation was performed as per the 3A assembly above but 4μl of plasmid DNA was used to 2μl of destination plasmid. </p>
-
<p>For the digestion we used Fermantas enzymes instead. The protocol remained the same but we used Ferantas Buffer H instead of the NEB Buffer 2. </p>
+
<p>For the digestion we used Fermentas enzymes instead. The protocol remained the same but we used Fermentas Buffer H instead of the NEB Buffer 2. </p><br>
-
<p>
+
-
</p>
+
-
<p>There was not enough time to transform any of these this afternoon so after the ligation they were stored at -20C for the weekend. </p>  
+
<p>There was not enough time to transform any of these this afternoon so after the ligation they were stored at -20°C for the weekend. </p>  
      
      
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    <p><u>Website Designed and Built by: Ryan Edginton, James Lynch & Alex Clowsley</u> &nbsp;&nbsp;|&nbsp;&nbsp;
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    <a href="https://igem.org/Team.cgi?id=764" style="color:#57B947" target="_blank"><u>Contact Us</u></a>  &nbsp;&nbsp;|&nbsp;&nbsp;
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    <a href="https://2012.igem.org/Team:Exeter/site_map" style="color:#57B947"><u>Site Map</u></a></p>
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Latest revision as of 00:07, 27 September 2012

ExiGEM2012 Lab Book 3GP wk6

The 3-Gene Inducible Plasmid: 10th - 14th September 2012

Monday 10th September

Morning

3A Assembly

BBa_K206000_BBaB0034 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (1)

BBa_K206000_BBaB0034 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (2)

BBa_J13002 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (1)

BBa_J13002 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (2)

BBa_J23119 + wbnJ BBa_B0014 in pSB1C3

BBa_B0034 + wfcA BBaB0014 in pSB1C3

BBa_B0034 + wfcA BBaB0014 in pSB1K3

BBaK094120_BBa_B0034 + wfcA BBaB0014 in pSB1C3

Performed with the NEB protocol with some adaptations again. The volume of digestion was changed from 50μl to 20μl by adding less water so as to increase the DNA concentration. The enzymes and buffer concentrations were adapted as below. This was not a linearized plasmid backbone so for the plasmid digestion we used the NEB Biolabs protocol adapted to the amounts shown below.


Tuesday 11th September

Morning

Transferring BioBricks into pSB1C3

wbnJ into pSB1C3

wbnK into pSB1C3

wfcA into pSB1C3

wbbC(1) into pSB1C3

wbbC(2) into pSB1C3

wbbC(3) into pSB1C3

wclY into pSB1C3

wbiP into pSB1C3

wbiP into pSB1C3

The genes and the destination plasmid were cut using EcoR1 and PstI. The digest was incubated for 90 minutes and then deactivated by heating at 80°C for 20 minutes. The ligation was performed as per the 3A assembly above but 4µl of plasmid DNA was used to 2µl of destination plasmid.


Afternoon

Transformation

The ligations above and the ligations from the 12/09/2012 were transformed using the Competent Cell protocol using our competent cells made in the lab.


Wednesday 12th September

Morning

wbbC(1) into pSB1C3, wbbC(2) into pSB1C3, wbbC(3) into pSB1C3 and wbiP into pSB1C3 did not form colonies on the plates. They were repeated.


Transferring BioBricks into pSB1C3

ompA into pSB1C3

wbbC(1) into pSB1C3

wbbC(2) into pSB1C3

wbbC(3) into pSB1C3

wbiP into pSB1C3

The genes and the destination plasmid were cut using EcoR1 and PstI. The digest was incubated for 90 minutes and then deactivated by heating at 80°C for 20 minutes. The ligation was performed as per the 3A assembly above but 4μl of plasmid DNA was used to 2μl of destination plasmid.


3A Assembly

ompA + amyA BBa_B0014

Performed with the NEB protocol with some adaptations again. The volume of digestion was changed from 50μl to 20μl by adding less water so as to increase the DNA concentration. The enzymes and buffer concentrations were adapted as below. This was not a linearized plasmid backbone so for the plasmid digestion we used the NEB Biolabs protocol adapted to the amounts shown below.


Two Step Phusion PCR using a 6 X 10 serial dilution

The reaction was set up using the Biolabs Phusion PCR protocol.


Two reactions were set up:

Reaction 1: Pbad Large and wbnJ using the primers supplied. See Operon Construction: 9th - 13th July 2012

Reaction 2: Reaction 1: Pbad Large and wbnJ using the primers supplied and varying the magnesium concentration. See Operon Construction: 9th - 13th July 2012

After PCR, PCR mix was dyed, loaded onto the gel and run for 20 minutes. No bands were shown on the gel so the PCR did not work.


Afternoon

Transferring colonies to liquid medium

Cells transformed with below were added into liquid medium and incubated overnight.

BBa_E0240

BBa_K206000_BBaB0034 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (1)

BBa_K206000_BBaB0034 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (2)

BBa_J13002 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (1)

BBa_J13002 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (2)

BBa_J23119 + wbnJ BBa_B0014 in pSB1C3

BBa_B0034 + wfcA BBaB0014 in pSB1C3

BBaK094120_BBa_B0034 + wfcA BBaB0014 in pSB1C3

wbnJ into pSB1C3

wbnK into pSB1C3

wfcA into pSB1C3

wclY into pSB1C3

wbiP into pSB1C3

Protocol was followed using chloramphenicol for the BioBrick part as the selection antibiotic.


Thursday 13th. September

Morning

ompA + amyA BBa_B0014 showed no colonies on the overnight plate.

Miniprepping

BBa_E0240

BBa_K206000_BBaB0034 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (1)

BBa_K206000_BBaB0034 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (2)

BBa_J13002 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (1)

BBa_J13002 + ompA BBa_K322921_BBa_B0014 in pSB1C3 (2)

BBa_J23119 + wbnJ BBa_B0014 in pSB1C3

BBa_B0034 + wfcA BBaB0014 in pSB1C3

BBaK094120_BBa_B0034 + WfcA BBaB0014 in pSB1C3

wbnJ into pSB1C3

wbnK into pSB1C3

wfcA into pSB1C3

wclY into pSB1C3

wbiP into pSB1C3

All mini-preps were nanodropped showing very good concentrations.


Afternoon

Gel Digest of DNA from 21/08/2012

A digestion using EcoR1 and PstI was run on all of the miniprepped DNA from today.

For the digestion we used Fermentas enzymes instead. The protocol remained the same but Fermentas Buffer H was used instead of the NEB Buffer 2.


Friday 14th September

Today a digestion/ligation was set up to put our remaining BioBricks into pSB1C3.

wbnJ

BBa_K094120_BBaB0034 in pSB1C3

wclY in pSB1C3

wbbc in pSB1C3

wfcA in pSB1C3

BBaB0034_wbiP in pSB1C3

BBaB0034_wbnK in pSB1C3

BBaB0034_wclY in pSB1C3

BBa_K206000_BBa_B0034 in pSB1C3

BBa_ K206001_BBa_B0034 in pSB1C3

BBa_I0500_BBa_B0034 in pSB1C3

BBa_J23119_ BBa_B0034 in pSB1C3

ompA in pSB1C3

ompA K322921_BBa_B0014 in pSB1C3

BBa_K094120_BBa_B0034_wclY BBa_B0014 in pSB1C3

BBa_J13002 _BBa_B0034_wclY BBa_B0014 in pSB1C3

BBa_J13002 amyA BBa_B0014 in pSB1C3

BBa_J13002 hasA BBa_B0014 in pSB1C3

The genes and the destination plasmid were cut using EcoR1 and PstI. The digest was incubated for 90 minutes and then deactivated by heating at 80°C for 20 minutes. The ligation was performed as per the 3A assembly above but 4μl of plasmid DNA was used to 2μl of destination plasmid.

For the digestion we used Fermentas enzymes instead. The protocol remained the same but we used Fermentas Buffer H instead of the NEB Buffer 2.


There was not enough time to transform any of these this afternoon so after the ligation they were stored at -20°C for the weekend.

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