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

From 2014.igem.org

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<ul>
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<li><b>pSB1C3-alsS-ilvC-ilvD-kivD</b></li>
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<li>Optimization of PCR conditions for coding sequence amplification</li>
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<ul>
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<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>
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<li>product amount was increased by lower annealing temperatures</li>
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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>
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<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>
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<li>product amount was increased by lower annealing temperatures</li>
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<ul>
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<li>54°C was identified as optimal annealing temperature</li>
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<li>90 seconds were identified as optimal elongation time</li>
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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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<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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<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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<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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<li><b>pSB1C3-alsS-ilvC-ilvD-kivD</b></li>
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<li><i>Dpn</i>I digest of template molecules in all purified PCR samples</li>
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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 06:45, 27 August 2014


July

  • pSB1C3-alsS-ilvC-ilvD-kivD
    • 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
  • pSB1C3-alsS-ilvC-ilvD-kivD
    • 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