Team:Goettingen/week19-2

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Language: <img height="20", src="http://www.patrickreinke.de/igem/eng.jpg"> English, <img height="20", src="http://www.patrickreinke.de/igem/deu.jpg"> <a href="https://2012.igem.org/Team:Goettingen/main_deu">Deutsch</a> <br>
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<td width="900" bordercolor="black" valign="top">
<h2><b>V09_03 </b></h2><br>
<h2><b>V09_03 </b></h2><br>
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<b>Quickchange of <i>fliC</i></b><br>
+
<b>Overlapping PCR of <i>fliC</i></b><br>
<ul>
<ul>
-
<li>Experiment: <br>Quick Change overlapping PCR was performed using designed primers and Pfu Turbo polymerase (see Quick Change protocol in "protocols").<br></li>
+
<li>Experiment: <br> In order to eliminate undesired restriction sites inside of our <i>fliC</i> part a Overlapping PCR was performed using especially designed primers that will cause a point mutation and <i>PfuTurbo</i> polymerase (<a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>). Since four unwanted restriction sites are localized in our part four reactions were prepared producing overlapping fragments. Subsequently samples 1 and 2 as well as 3 and 4 were combined via overlapping PCR reducing the number of fragments at two. Finally sample 1+2 and 3+4 were combined as well and one final DNA fragment is obtained. <br></li>
<li>Observations & Results: <br>
<li>Observations & Results: <br>
  The QC PCR was not successful. We were not able to observe any PCR product.</li>
  The QC PCR was not successful. We were not able to observe any PCR product.</li>
Line 549: Line 38:
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<td width="900" bordercolor="black" valign="top">
<h2><b>V09_04 </b></h2><br>
<h2><b>V09_04 </b></h2><br>
-
<b>V09_04_1 Test digestion of pSB1C3(including <i>flHDC</i> under the control of different promoters)</b><br>
+
<b>V09_04_1 Test digestion of pSB1C3 (including <i>flHDC</i> under the control of different promoters)</b><br>
<ul>
<ul>
-
<li>Experiment: <br>The test digestion of pSB1C3 including <i>flHDC</i> under the expression of 8 different promoters was performed with the restriction enzymes EcoRI and PstI according to the protocol. The product was subsequently analyzed on an 1% analytical agarose gel.<br> </li>
+
<li>Experiment: <br>The test digestion of pSB1C3 including <i>flHDC</i> under the expression of 8 different promoters was performed with the restriction enzymes <i>EcoRI</i> and <i>PstI</i> according to the <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>. The product was subsequently analyzed on an 1% analytical agarose gel.<br> </li>
<li>Observations & Results: <br>
<li>Observations & Results: <br>
Since fragments of the expected size could be obtained for all clones the test digestion worked for all.</li>
Since fragments of the expected size could be obtained for all clones the test digestion worked for all.</li>
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<b>V09_05_1 Preparative double digestion of <i>motA</i>, <i>motB</i>, <i>yhjH</i> and pSB1C3</b><br>
<b>V09_05_1 Preparative double digestion of <i>motA</i>, <i>motB</i>, <i>yhjH</i> and pSB1C3</b><br>
<ul>
<ul>
-
<li>Experiment: <br> <i>motA</i>, <i>motB</i>, <i>yhjH</i> and pSB1C3 were digested with EcoRI and PstI. The prodcuts were seperated via gel electrophoresis and purified using PeqGOLD Gelextraction Kit (Peqlab).</li>
+
<li>Experiment: <br> <i>motA</i>, <i>motB</i>, <i>yhjH</i> and pSB1C3 were digested with <i>EcoRI</i> and <i>PstI</i>. The prodcuts were seperated via gel electrophoresis and purified using PeqGOLD Gelextraction Kit (Peqlab).</li>
</ul>  
</ul>  
<br>
<br>
-
<b>V09_05_2 Quickchange of <i>fliC</i></b><br>
+
<b>V09_05_2 Repetition of the Overlapping PCR of <i>fliC</i></b><br>
<ul>
<ul>
-
<li>Experiment: <br> Quick Change overlapping PCR was performed using designed primers and Pfu Turbo polymerase (see Quick Change protocol in "protocols").<br></li>
+
<li>Experiment: <br> Here again the Overlapping PCR was performed using designed primers and <i>PfuTurbo</i> polymerase (<a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>). However, this time a lower annealing temperature was chosen. Also this time four short fragments were produced first and subsequently combined to a complete final DNA fragment. <br></li>
<li>Observations & Results: <br>
<li>Observations & Results: <br>
-
  The QC PCR was not successful again. We were not able to observe any PCR product.</li>
+
  Once again the QC PCR failed for no PCR product was observed. Since we already had problems with enzymes during our experiments we consider an non-functionable polymerase. </li>
</ul>
</ul>
<br></td></tr>
<br></td></tr>
</table>
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<table cellpadding="20 px" border="1" bordercolor="black" valign="top">
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<td width="900" bordercolor="black" valign="top">
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<h2><b>V09_06 </b></h2><br>
<h2><b>V09_06 </b></h2><br>
-
<b>Ligation and Transformation of <i>motA</i>, <i>motB</i> and <i>yhjH</i></b><br>
+
<b>V09_06_1  Ligation of <i>motA</i>, <i>motB</i> and <i>yhjH</i> into pSB1C3 </b><br>
<ul>
<ul>
-
<li>Experiment: <br>After the ligation of <i>motA</i>, <i>motB</i> and <i>yhjH</i> into the plasmid pSB1C3, the ligation products were transformed into the <i>E. coli</i> strain DH10B.</li>
+
<li>Experiment: <br>
 +
The ligation of <i>motA</i>, <i>motB</i> and <i>yhjH</i> with pSB1C3 was conducted as described in the <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>. <br></li>
</ul>
</ul>
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<b>V09_06_2 Chemical transformation of <i>motA</i>, <i>motB</i> and <i>yhjH</i> to <i>E. coli</i> DH10B</b><br>
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<li>Experiment:  <br>
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The transformation was performed as described in the standard protocol. <br>
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<li>Observations & Results: <br>
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The transformation was successful since numerous colonies has formed whereas the negative control remained clear.</li>
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<li>Experiment: <br>Over night cultures were prepared:<br>
<li>Experiment: <br>Over night cultures were prepared:<br>
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pSB1C3-<i>motA</i><br>
pSB1C3-<i>motA</i><br>
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pSB1C3-<i>motB</i></li>
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pSB1C3-<i>motB</i><br>
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pSB1C3-<i>yhjH</i><br>
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<b>Important pages</b>:<br>
 
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<a href="https://2012.igem.org/Team:Goettingen">Home</a>;
 
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Latest revision as of 17:18, 22 September 2012

Deutsch  / English 

#2 Speed Improvement - 19th week

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V09_03


Overlapping PCR of fliC
  • Experiment:
    In order to eliminate undesired restriction sites inside of our fliC part a Overlapping PCR was performed using especially designed primers that will cause a point mutation and PfuTurbo polymerase (protocol). Since four unwanted restriction sites are localized in our part four reactions were prepared producing overlapping fragments. Subsequently samples 1 and 2 as well as 3 and 4 were combined via overlapping PCR reducing the number of fragments at two. Finally sample 1+2 and 3+4 were combined as well and one final DNA fragment is obtained.
  • Observations & Results:
    The QC PCR was not successful. We were not able to observe any PCR product.


V09_04


V09_04_1 Test digestion of pSB1C3 (including flHDC under the control of different promoters)
  • Experiment:
    The test digestion of pSB1C3 including flHDC under the expression of 8 different promoters was performed with the restriction enzymes EcoRI and PstI according to the protocol. The product was subsequently analyzed on an 1% analytical agarose gel.
  • Observations & Results:
    Since fragments of the expected size could be obtained for all clones the test digestion worked for all.

V09_04_2 Biobrick Standardization of motA,motB and yhjH
  • Experiment:
    PCR of motA, motB and yhjH using primers containing the standard BioBrick sequences. Procedure was succesful and the PCR products were purified via PeqGOLD Gelextraction Kit (Peqlab).


V09_05


V09_05_1 Preparative double digestion of motA, motB, yhjH and pSB1C3
  • Experiment:
    motA, motB, yhjH and pSB1C3 were digested with EcoRI and PstI. The prodcuts were seperated via gel electrophoresis and purified using PeqGOLD Gelextraction Kit (Peqlab).

V09_05_2 Repetition of the Overlapping PCR of fliC
  • Experiment:
    Here again the Overlapping PCR was performed using designed primers and PfuTurbo polymerase (protocol). However, this time a lower annealing temperature was chosen. Also this time four short fragments were produced first and subsequently combined to a complete final DNA fragment.
  • Observations & Results:
    Once again the QC PCR failed for no PCR product was observed. Since we already had problems with enzymes during our experiments we consider an non-functionable polymerase.


V09_06


V09_06_1 Ligation of motA, motB and yhjH into pSB1C3
  • Experiment:
    The ligation of motA, motB and yhjH with pSB1C3 was conducted as described in the protocol.

V09_06_2 Chemical transformation of motA, motB and yhjH to E. coli DH10B
  • Experiment:
    The transformation was performed as described in the standard protocol.
  • Observations & Results:
    The transformation was successful since numerous colonies has formed whereas the negative control remained clear.


V09_09


Preparation of over night cultures
  • Experiment:
    Over night cultures were prepared:

    pSB1C3-motA
    pSB1C3-motB
    pSB1C3-yhjH



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