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

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 of pSB1C3</b></li>
 +
        <ul>
 +
  <li>We tried to redo the amplification of last week.</li>
 +
                  <ul>
 +
                      <li>Optimization of PCR conditions for coding sequence amplification</li>
 +
                      <ul>
 +
                        <li>combinations of primers and templates as <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Jun#Week5" target="_blank">described before</a></li>
 +
                        <li>annealing temperature gradients from 50°C to 58°C were tried</li>
 +
                        <li>product amount was increased by lower annealing temperatures</li>
 +
                      </ul>
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                  </ul>
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        </ul>
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            </ul>
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<ul>
 
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<li><b>pSB1C3-alsS-ilvC-ilvD-kivD</b></li>
 
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<ul>
 
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<li>Optimization of PCR conditions for coding sequence amplification</li>
 
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<ul>
 
-
<li>combinations of primers and templates as <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Jun#Week5" target="_blank">described before</a></li>
 
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<li>annealing temperature gradients from 50°C to 58°C were tried</li>
 
-
<li>product amount was increased by lower annealing temperatures</li>
 
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</ul>
 
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</ul>
 
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</ul>
 
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             </div>
             </div>
             <div class="content">
             <div class="content">
 +
            <ul>
 +
        <li><b><i>kivD</i>, <i>alsS</i>, <i>ilvC</i>, <i>ilvD</i> and backbone of pSB1C3</b></li>
 +
        <ul>
 +
  <li>We tried to redo the amplification of last week.</li>
 +
                  <ul>
 +
                      <li>Optimization of PCR conditions for coding sequence amplification</li>
 +
                      <ul>
 +
                        <li>combinations of primers and templates as <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Jun#Week5" target="_blank">described before</a></li>
 +
                        <li>annealing temperature gradients from 50°C to 58°C were tried</li>
 +
                        <li>product amount was increased by lower annealing temperatures</li>
 +
                        <ul>
 +
                            <li>54°C was identified as optimal annealing temperature</li>
 +
                            <li>90 seconds were identified as optimal elongation time</li>
 +
                        </ul>
 +
                      </ul>
 +
                  </ul>
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        </ul>
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            </ul>
         </div>
         </div>
       </div>
       </div>
     </div>
     </div>
     </div>
     </div>
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-
 
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<ul>
 
-
<li><b>pSB1C3-alsS-ilvC-ilvD-kivD</b></li>
 
-
<ul>
 
-
<li>Optimization of PCR conditions for coding sequence amplification</li>
 
-
<ul>
 
-
<li>combinations of primers and templates as <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Jun#Week5" target="_blank">described before</a></li>
 
-
<li>annealing temperature gradients from 50°C to 58°C were tried</li>
 
-
<li>product amount was increased by lower annealing temperatures</li>
 
-
<ul>
 
-
<li>54°C was identified as optimal annealing temperature</li>
 
-
<li>90 seconds were identified as optimal elongation time</li>
 
-
</ul>
 
-
</ul>
 
-
</ul>
 
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</ul>
 
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             <div class="content">
             <div class="content">
 +
            <ul>
 +
        <li><b><i>kivD</i>, <i>alsS</i>, <i>ilvC</i>, <i>ilvD</i></b></li>
 +
        <ul>
 +
  <li>With optimized conditions the amplification should give results this week</li>
 +
                  <ul>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> as <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Jun#Week5" target="_blank">described before</a></li>
 +
                      <li>PCR products were extracted out of the gel.</li>
 +
                      <li>Plasmid purfication of the constructs</li>
 +
                  <ul>
 +
        </ul>
 +
              <li><b>Backbone of pSB1C3</b></li>
 +
              <ul>
 +
                  <li>We aim to amplify the backbone with Q5 polymerase</li>
 +
                  <ul>
 +
                    <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_kivD_pSB1C3" target="_blank">fw_kivD_pSB1C3</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_alsS_pSB1C3" target="_blank">rv_alsS_pSB1C3</a>)</li>
 +
            <ul>
 +
        <li>Elongation time: ...</li>
 +
        <li>Bands (not) as expected (... bp)</li>
 +
            </ul>
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                    <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">PCR purification</a> of backbone</li>
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                  </ul>
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              </ul>
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            </ul>
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         </div>
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     </div>
     </div>
     </div>
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-
 
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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 coding sequences was repeated using <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/July#Week2" target="_blank">optimized conditions</a></li>
 
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<ul>
 
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<li>Gel extraction of PCR products using GeneJET Gel Extraction Kit from ThermoScientific </li>
 
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<li>PCR product prufication was carried out using GeneJET PCR Purification Kit from ThermoScientific</li>
 
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</ul>
 
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<li>pSB1C3 backbone was amplified using Q5 polymerase from NEB</li>
 
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<ul>
 
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<li>PCR products were analyzed by agarose gel electrophoresis</li>
 
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<li>PCR product purification by Wizard SV Gel and PCR Clean-Up System (Promega)</li>
 
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</ul>
 
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</ul>
 
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<ul>
 
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                 <a  style="font-size:24px" href="#"><p style="margin-left:42%">Week 4 &nbsp;&nbsp;&nbsp; 07/28 - 08/03</p></a>
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                 <h6><a  style="font-size:24px" href="#">Week 4 &nbsp;&nbsp;&nbsp; 07/28 - 08/03</a></h6>
             </div>
             </div>
             <div class="content">
             <div class="content">
 +
            <ul>
 +
        <li><b><i>kivD</i>, <i>alsS</i>, <i>ilvC</i>, <i>ilvD</i></b></li>
 +
        <ul>
 +
  <li>This week we aim to prove if the constructs were right</li>
 +
                  <ul>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <i>DpnI</i>...</li>
 +
              <ul>
 +
        <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>Transformation with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">electrocompotetent cells</a></li>
 +
                  <ul>
 +
        </ul>
 +
            </ul>
         </div>
         </div>
       </div>
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     </div>
     </div>
     </div>
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<ul>
 
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<li><b>pSB1C3-alsS-ilvC-ilvD-kivD</b></li>
 
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<ul>
 
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<li><i>Dpn</i>I digest of template molecules in all purified PCR samples</li>
 
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<ul>
 
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<li>1µL (10 units) of enzyme were used for 30 µL plasmid solution (about 3 µg of DNA) </li>
 
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<li>Incubation at 37°C for three hours</li>
 
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</ul>
 
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<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>
 
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<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>
 
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Revision as of 11:47, 27 August 2014


July

Week 1     07/07 - 07/13

Week 1     07/07 - 07/13
  • kivD, alsS, ilvC, ilvD and backbone of pSB1C3
    • We tried to redo the amplification of last week.
      • Optimization of PCR conditions for coding sequence amplification
        • combinations of primers and templates as described before
        • annealing temperature gradients from 50°C to 58°C were tried
        • product amount was increased by lower annealing temperatures
Week 2     07/14 - 07/20

Week 2     07/14 - 07/20
  • kivD, alsS, ilvC, ilvD and backbone of pSB1C3
    • We tried to redo the amplification of last week.
      • Optimization of PCR conditions for coding sequence amplification
        • combinations of primers and templates as described before
        • annealing temperature gradients from 50°C to 58°C were tried
        • product amount was increased by lower annealing temperatures
          • 54°C was identified as optimal annealing temperature
          • 90 seconds were identified as optimal elongation time
Week 3     07/21 - 07/27

Week 3     07/21 - 07/27
Week 4     07/28 - 08/03
Week 4     07/28 - 08/03

Retrieved from "http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Jul"