Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Aug

From 2014.igem.org

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              <ul>
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        <li><b><i>kivD</i>, <i>alsS</i>, <i>ilvC</i>, <i>ilvD</i> and backbone pSB1C3</b></li>
 +
        <ul>
 +
    <li>This week we tried to begin from zero again with optimized conditions and new competent cells.</li>
 +
                    <ul>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a>)</li>
 +
              <ul>
 +
                          <li>Primer were used as described before</li>
 +
                          <li>pSB1K3-RFP was used as template for backbone amplification</li>
 +
          <li>Annealing temperature: ...</li>
 +
          <li>Bands (not) as expected (... bp)</li>
 +
              </ul>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>DpnI</i></a></li>
 +
              <ul>
 +
                          <li>Aberation from protocol: Incubation for about 10 hours.</li>
 +
          <li>Bands (not) as expected (... bp)</li>
 +
              </ul>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with <i>kivD</i>, <i>alsS</i>, <i>ilvC</i> and  <i>ilvD</i> on pSB1C3</li>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells</li>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_ilvC_ilvD" target="_blank">fw_ilvC_ilvD</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_pSB1C3_kivD" target="_blank">rv_pSB1C3_kivD</a>)</li>
 +
              <ul>
 +
          <li>Annealing temperature: 65°C</li>
 +
          <li>Bands as expected (3,6 kb)</li>
 +
              </ul>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_ilvC" target="_blank">fw_alsS_ilvC</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_ilvD_ilvC" target="_blank">rv_ilvD_ilvC</a>)</li>
 +
              <ul>
 +
          <li>Annealing temperature: 65°C</li>
 +
          <li>Bands as expected (3,3 kb)</li>
 +
              </ul>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1C3-alsS-ilvC-ilvD-kivD</li>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>NotI</i></a></li>
 +
              <ul>
 +
                  <li>Bands as expected (backbone: 2,2 kb and insert: 6,7 kb)</li>
 +
              </ul>
 +
                      <li>A glycerin stock was created for positive clones</li>
 +
                    </ul>
 +
        </ul>
 +
              </ul>
         </div>
         </div>
       </div>
       </div>
     </div>
     </div>
     </div>
     </div>
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<ul>
 
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<li><b>pSB1C3-alsS-ilvC-ilvD-kivD</b></li>
 
-
<ul>
 
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<li>Amplification of all five parts was repeated with Q5 polymerase from NEB</li>
 
-
<ul>
 
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<li>PCR conditions were set as used before</li>
 
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<li><i>pSB1K3-RFP</i> was used as template for pSB1K3 backbone amplification (kanamycin resistance was choosen to have another selection marker)</li>
 
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</ul>
 
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<li>PCR products were purified using Wizard® SV Gel an PCR Clean-Up System (Promega) </li>
 
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<li><i>Dpn</i>I digest of template molecules in purified PCR products of the backbone</li>
 
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<ul>
 
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<li>3µL (30 units) of <i>Dpn</i>I (Fermentas) were used for 30 µL plasmid solution (about 3 µg of DNA) </li>
 
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<li>Incubation at 37°C for about ten hours</li>
 
-
</ul>
 
-
 
-
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with all four coding sequences (<i>alsS</i>, <i>ilvC</i>, <i>ilvD</i>, <i>kivD</i>) and <i>pSB1C3</i></li>
 
-
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation of electrocompetent <i>E. coli</i> KRX cells</a></li>
 
-
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_ilvC_ilvD" target="_blank">fw_ilvC_ilvD</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_pSB1C3_kivD" target="_blank">rv_pSB1C3_kivD</a>)</li>
 
-
<ul>
 
-
<li>Annealing temperature was set to 65°C to achieve strict conditions</li>
 
-
<li>Agarose gel electrophoresis showed products of expected size (about 3.6kb)</li>
 
-
</ul>
 
-
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_alsS_ilvC" target="_blank">fw_alsS_ilvC</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_ilvD_ilvC" target="_blank">rv_ilvD_ilvC</a>)</li>
 
-
<ul>
 
-
<li>Annealing temperature was set to 65°C to achieve strict conditions</li>
 
-
<li>Agarose gel electrophoresis showed products of expected size (about 3.3kb)</li>
 
-
</ul>
 
-
<li>Positive clones were used to start liquid cultures</li>
 
-
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of <i>pSB1K3-alsS-ilvC-ilvD-kivD</i></li>
 
-
<li><i>Not</i>I digestion of isolated plasmids</li>
 
-
<ul>
 
-
<li>Components (10µL total volume):</li>
 
-
<ul>
 
-
<li>0.3µL <i>Not</i>I (Fermentas)</li>
 
-
<li>1µL 10 fold Organge buffer (Fermentas)</li>
 
-
<li>3µL plasmid solution (< µg of DNA)</li>
 
-
<li>5.7µL water</li>
 
-
</ul>
 
-
<li>Incubation at 37°C for one hour</li>
 
-
<li>Agarose gel electrophoresis showed expected fragment sizes:</li>
 
-
<ul>
 
-
<li>2.2kb - backbone</li>
 
-
<li>6.7kb - insert</li>
 
-
</ul>
 
-
</ul>
 
-
<li>Glycerin stocks for positive clones created</li>
 
-
<li>Sanger sequencing using <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VF-Primer" target="_blank">VF</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VR-Primer" target="_blank">VR</a> as sequencing primers</li>
 
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</ul>
 
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</ul>
 
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Revision as of 11:21, 28 August 2014


August

Week 1     08/04 - 08/10

Week 1     08/04 - 08/10
  • kivD, alsS, ilvC and ilvD
Week 2     08/11 - 08/17
Week 2     08/11 - 08/17
Week 3     08/18 - 08/24

Week 3     08/18 - 08/24
  • kivD, alsS, ilvC, ilvD and backbone pSB1C3

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