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

From 2014.igem.org

(Difference between revisions)
(cloning experiments added)
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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><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_kivD_ilvD" target="_blank">rv_kivD_ilvD</a>)</li>
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<ul>
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<li>Annealing temperature was set to 65°C to achieve strict conditions</li>
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<li>Agarose gel electrophoresis showed no products of expected size</li>
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</ul>
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</ul>
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</ul>
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     </div>
     </div>
     </div>
     </div>
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 +
 +
 +
 +
 +
 +
 +
 +
<ul>
 +
<li><b>pSB1C3-alsS-ilvC-ilvD-kivD</b></li>
 +
<ul>
 +
<li>Amplification of all five parts was repeated with Q5 polymerase from NEB</li>
 +
<ul>
 +
<li>PCR conditions were set as used before</li>
 +
<li><i>pSB1C3-RFP</i> was used as template for pSB1C3 backbone amplification</li>
 +
</ul>
 +
<li>PCR products were purified using Wizard® SV Gel an PCR Clean-Up System (Promega) </li>
 +
<li><i>Dpn</i>I digest of template molecules in all purified PCR samples</li>
 +
<ul>
 +
<li>1µL (10 units) of <i>Dpn</i>I (Fermentas) were used for 30 µL plasmid solution (about 3 µg of DNA) </li>
 +
<li>Incubation at 37°C for three 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_alsS_ilvC" target="_blank">fw_alsS_ilvC</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_kivD_ilvD" target="_blank">rv_kivD_ilvD</a>)</li>
 +
<ul>
 +
<li>Annealing temperature was set to 65°C to achieve strict conditions</li>
 +
<li>Agarose gel electrophoresis showed no products of expected size</li>
 +
</ul>
 +
</ul>
 +
</ul>
 +
 +
 +
 +
 +
 +
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Line 85: Line 153:
     </div>
     </div>
     </div>
     </div>
 +
 +
 +
 +
 +
 +
 +
 +
 +
<ul>
 +
<li><b>pSB1C3-alsS-ilvC-ilvD-kivD</b></li>
 +
<ul>
 +
<li>Amplification of all five parts was repeated with Q5 polymerase from NEB</li>
 +
<ul>
 +
<li>PCR conditions were set as used before</li>
 +
<li><i>pSB1K3-RFP</i> was used as template for pSB1K3 backbone amplification (kanamycin resistance was choosen to have another selection marker)</li>
 +
</ul>
 +
<li>PCR products were purified using Wizard® SV Gel an PCR Clean-Up System (Promega) </li>
 +
<li><i>Dpn</i>I digest of template molecules in purified PCR products of the backbone</li>
 +
<ul>
 +
<li>3µL (30 units) of <i>Dpn</i>I (Fermentas) were used for 30 µL plasmid solution (about 3 µg of DNA) </li>
 +
<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 06:49, 27 August 2014


August

  • pSB1C3-alsS-ilvC-ilvD-kivD
    • Colony PCR (fw_alsS_ilvC, rv_kivD_ilvD)
      • Annealing temperature was set to 65°C to achieve strict conditions
      • Agarose gel electrophoresis showed no products of expected size
  • pSB1C3-alsS-ilvC-ilvD-kivD
    • Amplification of all five parts was repeated with Q5 polymerase from NEB
      • PCR conditions were set as used before
      • pSB1C3-RFP was used as template for pSB1C3 backbone amplification
    • PCR products were purified using Wizard® SV Gel an PCR Clean-Up System (Promega)
    • DpnI digest of template molecules in all purified PCR samples
      • 1µL (10 units) of DpnI (Fermentas) were used for 30 µL plasmid solution (about 3 µg of DNA)
      • Incubation at 37°C for three hours
    • Gibson Assembly with all four coding sequences (alsS, ilvC, ilvD, kivD) and pSB1C3
    • Transformation of electrocompetent E. coli KRX cells
    • Colony PCR (fw_alsS_ilvC, rv_kivD_ilvD)
      • Annealing temperature was set to 65°C to achieve strict conditions
      • Agarose gel electrophoresis showed no products of expected size
  • pSB1C3-alsS-ilvC-ilvD-kivD
    • Amplification of all five parts was repeated with Q5 polymerase from NEB
      • PCR conditions were set as used before
      • pSB1K3-RFP was used as template for pSB1K3 backbone amplification (kanamycin resistance was choosen to have another selection marker)
    • PCR products were purified using Wizard® SV Gel an PCR Clean-Up System (Promega)
    • DpnI digest of template molecules in purified PCR products of the backbone
      • 3µL (30 units) of DpnI (Fermentas) were used for 30 µL plasmid solution (about 3 µg of DNA)
      • Incubation at 37°C for about ten hours
    • Gibson Assembly with all four coding sequences (alsS, ilvC, ilvD, kivD) and pSB1C3
    • Transformation of electrocompetent E. coli KRX cells
    • Colony PCR (fw_ilvC_ilvD, rv_pSB1C3_kivD)
      • Annealing temperature was set to 65°C to achieve strict conditions
      • Agarose gel electrophoresis showed products of expected size (about 3.6kb)
    • Colony PCR (fw_alsS_ilvC, rv_ilvD_ilvC)
      • Annealing temperature was set to 65°C to achieve strict conditions
      • Agarose gel electrophoresis showed products of expected size (about 3.3kb)
    • Positive clones were used to start liquid cultures
    • Plasmid isolation of pSB1K3-alsS-ilvC-ilvD-kivD
    • NotI digestion of isolated plasmids
      • Components (10µL total volume):
        • 0.3µL NotI (Fermentas)
        • 1µL 10 fold Organge buffer (Fermentas)
        • 3µL plasmid solution (< µg of DNA)
        • 5.7µL water
      • Incubation at 37°C for one hour
      • Agarose gel electrophoresis showed expected fragment sizes:
        • 2.2kb - backbone
        • 6.7kb - insert
    • Glycerin stocks for positive clones created
    • Sanger sequencing using VF and VR as sequencing primers

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