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

From 2014.igem.org

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             <ul>
             <ul>
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        <li><b><i>alsS</i>, <i>ilvC</i>, <i>ilvD</i>, <i>kivD</i> and backbone of pSB1C3</b></li>
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        <li><b><i>alsS</i>, <i>ilvC</i>, <i>ilvD</i>, <i>kivD</i> and backbone pSB1C3</b></li>
        <ul>
        <ul>
  <li>We tried to redo the amplification of last week.</li>
  <li>We tried to redo the amplification of last week.</li>
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             <ul>
             <ul>
-
        <li><b><i>alsS</i>, <i>ilvC</i>, <i>ilvD</i>, <i>kivD</i> and backbone of pSB1C3</b></li>
+
        <li><b><i>alsS</i>, <i>ilvC</i>, <i>ilvD</i>, <i>kivD</i> and backbone pSB1C3</b></li>
        <ul>
        <ul>
  <li>We tried to redo the amplification of last week.</li>
  <li>We tried to redo the amplification of last week.</li>
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                 </ul>
                 </ul>
        </ul>
        </ul>
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               <li><b>Backbone of pSB1C3</b></li>
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               <li><b>Backbone pSB1C3</b></li>
               <ul>
               <ul>
                   <li>We aim to amplify it with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#Polymerases" target="_blank">Q5 polymerase</a></li>
                   <li>We aim to amplify it with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#Polymerases" target="_blank">Q5 polymerase</a></li>
                   <ul>
                   <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>
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                     <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>) (Elongationtime: 60 sec)</li>
            <ul>
            <ul>
-
        <li>Elongation time: 90 s</li>
+
        <li>Annealing temperature: 54 °C</li>
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        <li>Bands not as expected (2,2 kb)</li>
+
        <li>Bands not as expected (~ 2.200 bp)</li>
            </ul>
            </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>
+
                     <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>) (Elongationtime: 80 sec)</li>
            <ul>
            <ul>
-
        <li>Elongation time: 80 s</li>
+
        <li>Annealing temperature: 54 °C</li>
-
        <li>Bands as expected (2,2 kb)</li>
+
        <li>Bands as expected (~ 2.200 bp)</li>
            </ul>
            </ul>
                     <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">PCR purification</a> of backbone</li>
                     <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">PCR purification</a> of backbone</li>

Latest revision as of 16:00, 16 October 2014


July

  • alsS, ilvC, ilvD, kivD and backbone 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, but still not good enough for Gibson
  • alsS, ilvC, ilvD, kivD and backbone 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
          • For the annealing temperature we identified 54°C as optimal.
          • For the elongation time 90 seconds worked better than 60 seconds.