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

From 2014.igem.org

(Difference between revisions)
(cloning experiments added)
 
(20 intermediate revisions not shown)
Line 40: Line 40:
<div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">  
<div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">  
-
<div id="text">
+
<div id="text" style="text-align:left">
<div class="tab" id="Week1">
<div class="tab" id="Week1">
             <div class="show">
             <div class="show">
Line 46: Line 46:
             </div>
             </div>
             <div class="hide">
             <div class="hide">
-
                 <a  style="font-size:24px" href="#"><p style="margin-left:42%">Week 1 &nbsp;&nbsp;&nbsp; 07/07 - 07/13</p></a>
+
                 <h6><a  style="font-size:24px" href="#">Week 1 &nbsp;&nbsp;&nbsp; 07/07 - 07/13</a></h6>
             </div>
             </div>
-
             <div class="content">
+
             <div class="content" style="margin-right:10%; margin-left:10%">
 +
            <ul>
 +
        <li><b><i>alsS</i>, <i>ilvC</i>, <i>ilvD</i>, <i>kivD</i> and backbone 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, but still not good enough for Gibson</li>
 +
                      </ul>
 +
                  </ul>
 +
        </ul>
 +
            </ul>
         </div>
         </div>
       </div>
       </div>
     </div>
     </div>
     </div>
     </div>
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
<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>
 
-
</ul>
 
-
</ul>
 
-
 
-
 
-
 
-
 
-
 
Line 84: Line 72:
<div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">  
<div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">  
-
   <div id="text">
+
   <div id="text" style="text-align:left">
<div class="tab" id="Week2">
<div class="tab" id="Week2">
             <div class="show">
             <div class="show">
Line 90: Line 78:
             </div>
             </div>
             <div class="hide">
             <div class="hide">
-
                 <a  style="font-size:24px" href="#"><p style="margin-left:42%">Week 2 &nbsp;&nbsp;&nbsp; 07/14 - 07/20</p></a>
+
                 <h6><a  style="font-size:24px" href="#">Week 2 &nbsp;&nbsp;&nbsp; 07/14 - 07/20</a></h6>
             </div>
             </div>
-
             <div class="content">
+
             <div class="content" style="margin-right:10%; margin-left:10%">
 +
            <ul>
 +
        <li><b><i>alsS</i>, <i>ilvC</i>, <i>ilvD</i>, <i>kivD</i> and backbone 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>For the annealing temperature we identified 54°C as optimal.</li>
 +
                            <li>For the elongation time 90 seconds worked better than 60 seconds.</li>
 +
                        </ul>
 +
                      </ul>
 +
                  </ul>
 +
        </ul>
 +
            </ul>
         </div>
         </div>
       </div>
       </div>
     </div>
     </div>
     </div>
     </div>
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
<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>
 
-
</ul>
 
-
 
-
 
-
 
-
 
-
 
-
 
Line 132: Line 110:
<div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">  
<div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">  
-
   <div id="text">
+
   <div id="text" style="text-align:left">
<div class="tab" id="Week3">
<div class="tab" id="Week3">
             <div class="show">
             <div class="show">
Line 138: Line 116:
             </div>
             </div>
             <div class="hide">
             <div class="hide">
-
                 <a  style="font-size:24px" href="#"><p style="margin-left:42%">Week 3 &nbsp;&nbsp;&nbsp; 07/21 - 07/27</p></a>
+
                 <h6><a  style="font-size:24px" href="#">Week 3 &nbsp;&nbsp;&nbsp; 07/21 - 07/27</a></h6>
             </div>
             </div>
-
             <div class="content">
+
             <div class="content" style="margin-right:10%; margin-left:10%">
 +
            <ul>
 +
        <li><b><i>alsS</i>, <i>ilvC</i>, <i>ilvD</i>, <i>kivD</i></b></li>
 +
        <ul>
 +
  <li>With the optimized conditions the amplifications were tried again.</li>
 +
                  <ul>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> with the optimized conditions and the primer combinations 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><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">Purfication</a> of the gel slices </li>
 +
                      <li>This worked well for <i>ilvC</i>, but not for <i>alsS</i>, <i>ilvD</i> and <i>kivD</i>.</li>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> with the optimized conditions and the primer combinations as <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Jun#Week5" target="_blank">described before</a> for <i>alsS</i>, <i>ilvD</i> and <i>kivD</i></li> 
 +
                      <li>PCR products were extracted out of the gel.</li>
 +
                      <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">Purfication</a> of the gel slices </li>
 +
                </ul>
 +
        </ul>
 +
              <li><b>Backbone pSB1C3</b></li>
 +
              <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>
 +
                  <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>) (Elongationtime: 60 sec)</li>
 +
            <ul>
 +
        <li>Annealing temperature: 54 °C</li>
 +
        <li>Bands not as expected (~ 2.200 bp)</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>) (Elongationtime: 80 sec)</li>
 +
            <ul>
 +
        <li>Annealing temperature: 54 °C</li>
 +
        <li>Bands as expected (~ 2.200 bp)</li>
 +
            </ul>
 +
                    <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">PCR purification</a> of backbone</li>
 +
                  </ul>
 +
              </ul>
 +
            </ul>
 +
 
         </div>
         </div>
       </div>
       </div>
     </div>
     </div>
     </div>
     </div>
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
-
<ul>
 
-
<li><b>pSB1C3-alsS-ilvC-ilvD-kivD</b></li>
 
-
<ul>
 
-
<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>
 
-
<ul>
 
-
<li>Gel extraction of PCR products using GeneJET Gel Extraction Kit from ThermoScientific </li>
 
-
<li>PCR product prufication was carried out using GeneJET PCR Purification Kit from ThermoScientific</li>
 
-
</ul>
 
-
<li>pSB1C3 backbone was amplified using Q5 polymerase from NEB</li>
 
-
<ul>
 
-
<li>PCR products were analyzed by agarose gel electrophoresis</li>
 
-
<li>PCR product purification by Wizard SV Gel and PCR Clean-Up System (Promega)</li>
 
-
</ul>
 
-
</ul>
 
-
<ul>
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
Line 181: Line 162:
<div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">  
<div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">  
-
   <div id="text">
+
   <div id="text" style="text-align:left">
<div class="tab" id="Week4">
<div class="tab" id="Week4">
             <div class="show">
             <div class="show">
Line 187: Line 168:
             </div>
             </div>
             <div class="hide">
             <div class="hide">
-
                 <a  style="font-size:24px" href="#"><p style="margin-left:42%">Week 4 &nbsp;&nbsp;&nbsp; 07/28 - 08/03</p></a>
+
                 <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" style="margin-right:10%; margin-left:10%">
 +
            <ul>
 +
        <li><b><i>alsS</i>, <i>ilvC</i>, <i>ilvD</i>, <i>kivD</i></b></li>
 +
        <ul>
 +
  <li>This week we aim to combine the four CDS's with the pSB1C3 backbone.</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> and pSB1C3</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>DpnI</i></a></li>
 +
                      <li>Transformation with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">electrocompetent</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaheatshock" target="_blank"> chemocompetent</a> cells</li>
 +
                  <ul>
 +
        </ul>
 +
            </ul>
         </div>
         </div>
       </div>
       </div>
     </div>
     </div>
     </div>
     </div>
-
 
-
 
-
 
-
 
-
 
-
<ul>
 
-
<li><b>pSB1C3-alsS-ilvC-ilvD-kivD</b></li>
 
-
<ul>
 
-
<li><i>Dpn</i>I digest of template molecules in all purified PCR samples</li>
 
-
<ul>
 
-
<li>1µL (10 units) of enzyme 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>
 
-
</ul>
 
-
</ul>
 
-
 
-
 
-
 
-
 
-
 
-
 
-
 
</html>
</html>

Latest revision as of 16:00, 16 October 2014


July

Week 1     07/07 - 07/13
Week 1     07/07 - 07/13
  • 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
Week 2     07/14 - 07/20
Week 2     07/14 - 07/20
  • 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.
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"