Team:Goettingen/week19-2

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                                <!-- <li><a href="https://2012.igem.org/Team:Goettingen/Human_Practice/Sponsors"><span><span>Sponsors</span></span></a></li>
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                                <!-- Text body -->
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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>
-
<b>Quikchange of <i>fliC</i></b><br>
+
<b>Overlapping PCR of <i>fliC</i></b><br>
<ul>
<ul>
-
<li>Experiment: <br> In order to eliminate undesired restriction sites inside of our part a Quikchange PCR was performed using especially designed primers that will cause a point mutation and Pfu Turbo polymerase (see Quick Change protocol in "protocols"). 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 smaple 1+2 and 3+4 were combined as well and one DNA fragment is obtained. <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>
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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>
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<td width="900" bordercolor="black" valign="top">
<h2><b>V09_06 </b></h2><br>
<h2><b>V09_06 </b></h2><br>
-
<b>V09_06_1 Insertion of <i>motA</i>, <i>motB</i> and <i>yhjH</i> into pSB1C3 </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>
<li>Experiment: <br>
-
The ligation of <i>motA</i>, <i>motB</i> and <i>yhjH</i> with pSB1C3 was conducted as described in the protocol. <br></li>
+
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>
<br>
<br>
Line 613: Line 102:
<ul>
<ul>
<li>Experiment: <br>Over night cultures were prepared:<br>
<li>Experiment: <br>Over night cultures were prepared:<br>
 +
<br>
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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Latest revision as of 17:18, 22 September 2012

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#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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