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

From 2014.igem.org

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              <ul>
              <ul>
        <li>Annealing temperature: 65 &deg;C</li>
        <li>Annealing temperature: 65 &deg;C</li>
-
        <li>Bands not as expected (3,45 kb) </li>
+
        <li>Bands not as expected (~ 3450 bp) </li>
              </ul>
              </ul>
                   </ul>
                   </ul>
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              <ul>
              <ul>
        <li>Annealing temperature: 65 &deg;C</li>
        <li>Annealing temperature: 65 &deg;C</li>
-
        <li>Bands not as expected (3,45 kb) </li>
+
        <li>Bands not as expected (~ 3450 bp) </li>
              </ul>
              </ul>
                   <ul>
                   <ul>
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                       <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (as described before)</li>
                       <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (as described before)</li>
              <ul>
              <ul>
-
                         <li>We used pSB1C3-RFP instead of pSB1C3-CFP as template for backbone amplification</li>
+
                         <li>We used pSB1C3-RFP (<a href="http://parts.igem.org/Part:BBa_J04450" target="_blank">J04450</a>) instead of pSB1C3-CFP as template for backbone amplification</li>
-
        <li>Elongation time: 80 s</li>
+
        <li>Annealing temperature: 65 °C</li>
-
        <li>Bands as expected (2,2 kp)</li>
+
        <li>Bands as expected (~ 2200 bp)</li>
              </ul>
              </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" target="_blank"><i>DpnI</i></a></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" target="_blank"><i>DpnI</i></a></li>
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              <ul>
              <ul>
        <li>Annealing temperature: 65 &deg;C</li>
        <li>Annealing temperature: 65 &deg;C</li>
-
        <li>Bands not as expected (3,45 bp)</li>
+
        <li>Bands not as expected (~ 3450 bp)</li>
              </ul>
              </ul>
                   </ul>
                   </ul>
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                           <li>pSB1K3-RFP was used as template for backbone amplification</li>
                           <li>pSB1K3-RFP was used as template for backbone amplification</li>
          <li>Annealing temperature: 53 &deg;C</li>
          <li>Annealing temperature: 53 &deg;C</li>
-
          <li>Bands as expected for <br><i>alsS</i>: about 1.8 kb, <br><i>ilvC</i>: about  1.55 kb, <br><i>ilvD</i>: about 1.95 kb, <br><i>kivD</i>: about 1.7 kb, <br>but not for pSB1K3-RFP</li>
+
          <li>Bands as expected for <br><i>alsS</i>: about 1800 bp, <br><i>ilvC</i>: about  1550 bp, <br><i>ilvD</i>: about 1950 bp, <br><i>kivD</i>: about 1700 bp, <br>but not for pSB1K3-RFP</li>
              </ul>
              </ul>
                       <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a></li> with Q5 for pSB1K3-RFP
                       <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a></li> with Q5 for pSB1K3-RFP
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                           <li>Primer were used as described before</li>
                           <li>Primer were used as described before</li>
          <li>Annealing temperature: 53 &deg;C</li>
          <li>Annealing temperature: 53 &deg;C</li>
-
          <li>Bands as expected (about 2.2 kb)</li>
+
          <li>Bands as expected (~ 2200 bp)</li>
              </ul>
              </ul>
                        
                        
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              <ul>
              <ul>
          <li>Annealing temperature: 65 &deg;C</li>
          <li>Annealing temperature: 65 &deg;C</li>
-
          <li>Bands as expected (3,6 kb)</li>
+
          <li>Bands as expected (~ 3600 bp)</li>
              </ul>
              </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>
                       <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>
              <ul>
          <li>Annealing temperature: 65 &deg;C</li>
          <li>Annealing temperature: 65 &deg;C</li>
-
          <li>Bands as expected (3,3 kb)</li>
+
          <li>Bands as expected (~ 3300 bp)</li>
              </ul>
              </ul>
<li>Liquid cultures for a restriction digest were prepared.</li>
<li>Liquid cultures for a restriction digest were prepared.</li>
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                       <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#RestrictionDigestion" target="_blank"><i>NotI</i></a></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#RestrictionDigestion" target="_blank"><i>NotI</i></a></li>
              <ul>
              <ul>
-
                  <li>Bands as expected (backbone: 2,2 kb and insert: 7 kb)</li>
+
                  <li>Bands as expected (backbone:~ 2200 bp and insert: ~ 7000 bp)</li>
              </ul>
              </ul>
                     </ul>
                     </ul>
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  <li>This week the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#L.lactis" target="_blank"> <i>Lactococcus lactis</i></a> arrived. We tried to amplify it's <i>adhA</i>.</li>
  <li>This week the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#L.lactis" target="_blank"> <i>Lactococcus lactis</i></a> arrived. We tried to amplify it's <i>adhA</i>.</li>
                   <ul>
                   <ul>
-
                    PCR:
+
                  <ul>
 +
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAisolation" target="_blank">DNA isolation</a> of <i>Lactococcus lactis</i></li>
 +
</ul>
 +
 
<ul>
<ul>
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> of the <i>adhA</i> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rev-pBS1C3-adhA" target="_blank">rev-pBS1C3-adhA</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw-pSB1C3_adhA" target="_blank">fw-pSB1C3_adhA</a>) and the Backbone out of psB1K3-<i>RFP</i> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rev-adhA_pSB1C3" target="_blank">rev-adhA_pSB1C3</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw-adhA-pSB1C3" target="_blank">fw-adhA-pSB1C3</a>)</li>
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> of the <i>adhA</i> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rev-pBS1C3-adhA" target="_blank">rev-pBS1C3-adhA</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw-pSB1C3_adhA" target="_blank">fw-pSB1C3_adhA</a>) and the Backbone out of psB1K3-<i>RFP</i> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rev-adhA_pSB1C3" target="_blank">rev-adhA_pSB1C3</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw-adhA-pSB1C3" target="_blank">fw-adhA-pSB1C3</a>)</li>
<ul>
<ul>
<li>Annealing temperature: 54 &deg;C</li>
<li>Annealing temperature: 54 &deg;C</li>
-
<li>Bands as expected (~1.2 kb for the <i>adhA</i> and 2.2 kb for the Backbone)</li>
+
<li>Bands as expected (~1200 bp for the <i>adhA</i> and ~ 2200 bp for the Backbone)</li>
</ul>
</ul>
          
          
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</ul>
</ul>
<ul>
<ul>
-
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with <i>[Part1]</i> and [Backbone]</li>
+
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson Assembly</a> with <i>adhA</i> and pSB1K3</li>
</ul>
</ul>
<ul>
<ul>
Line 226: Line 229:
<ul>
<ul>
<li>Annealing temperature: 65°C</li>
<li>Annealing temperature: 65°C</li>
-
<li>Bands as expected (~1.2 kb)</li>
+
<li>Bands as expected (~ 1200 bp)</li>
</ul>
</ul>
</ul>
</ul>
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<ul>
<ul>
<li>Annealing temperature: 65 °C</li>
<li>Annealing temperature: 65 °C</li>
-
<li>Bands as expected (~ 1.8 bp)</li>
+
<li>Bands as expected (~ 1800 bp)</li>
</ul>
</ul>
</ul>
</ul>

Latest revision as of 16:49, 16 October 2014


August

  • alsS, ilvC, ilvD, kivD and backbone pSB1K3
    • This week we tried to begin from zero again with optimized conditions and new competent cells.
      Therefor we prepared new plasmids from alsS, ilvC, ilvD, kivD and the backbone pSB1K3-RFP