Team:Goettingen/week6-2

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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>V06_04 </b></h2><br>
<h2><b>V06_04 </b></h2><br>
-
<b>Chemical retransformartion of the <i>flhDC</i>-promoter constructs into <i>E. coli</i> (DH10B)</i></b><br>
+
<b>Chemical retransformation of the <i>flhDC</i>-promoter constructs into <i>E. coli</i> (DH10B)</b><br>
<ul>
<ul>
<li>Experiment: <br>  
<li>Experiment: <br>  
-
For the chemical retransformation the standard protocol for transformation was followed. The following constructs were transformed into
+
For the chemical retransformation the standard <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a> for transformation was followed. The following constructs were transformed into
E. coli: <br>
E. coli: <br>
 +
<br>
20E - #2 <br>
20E - #2 <br>
20E - #3 <br>
20E - #3 <br>
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18M - #3 <br>
18M - #3 <br>
18C - #1 <br>
18C - #1 <br>
-
18C - #2 <br></li>  
+
18C - #2 <br>
 +
<br></li>  
<li>Observations & Results: <br>
<li>Observations & Results: <br>
-
The retransformation was successful since all plates showed numeours colonoes except the negative control.
+
The retransformation was successful since all plates showed numeours colonies except the negative control.
<br> </li>
<br> </li>
</ul>
</ul>
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<td width="900" bordercolor="black" valign="top">
<td width="900" bordercolor="black" valign="top">
<h2><b>V06_05 </b></h2><br>
<h2><b>V06_05 </b></h2><br>
-
<b>V06_05_1 Preparative double digestion of <i>flhDC</i> and plasmids containing further promoter</b><br>
+
<b>V06_05_1 Preparative double digestion of <i>flhDC</i> and plasmids containing further promoters</b><br>
<ul>
<ul>
<li>Experiment:  <br>
<li>Experiment:  <br>
-
In order to clone <i>flhDC</i> behind further promoters, the purified PCR product was digested with XbaI and PstI, whereas the BioBrick plasmids were cut with SpeI and PstI. The digestion was performed as described in the protocol. This time the follwing promoters were used: <br>
+
In order to clone <i>flhDC</i> behind further promoters, the purified PCR product was digested with <i>XbaI</i> and <i>PstI</i>, whereas the BioBrick plasmids were cut with <i>SpeI</i> and <i>PstI</i>. The digestion was performed as described in the <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>l. This time the follwing promoters were used: <br>
 +
<br>
J23113 - 20G - x21 <br>
J23113 - 20G - x21 <br>
J23109  - 2G - x106 <br>
J23109  - 2G - x106 <br>
Line 563: Line 55:
J23106 - 18O - x1185 <br>
J23106 - 18O - x1185 <br>
J23104 - 18K - x 1831 <br>
J23104 - 18K - x 1831 <br>
-
 
+
<br>
</ul>
</ul>
<br>
<br>
Line 569: Line 61:
<ul>
<ul>
<li>Experiment:  <br>
<li>Experiment:  <br>
-
The ligation of the digested flhDC with the BioBrick plasmids was conducted as described in the protocol. <br> </li>
+
The ligation of the digested <i>flhDC</i> with the BioBrick plasmids was conducted as described in the <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>. <br> </li>
</ul>
</ul>
<br></td></tr>
<br></td></tr>
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<td width="900" bordercolor="black" valign="top">
<td width="900" bordercolor="black" valign="top">
<h2><b>V06_06 </b></h2><br>
<h2><b>V06_06 </b></h2><br>
-
<b>Chemical transformartion of the <i>flhDC</i>-promoter constructs into <i>E. coli</i> (DH10B)</i></b><br>
+
<b>Chemical transformation of the <i>flhDC</i>-promoter constructs into <i>E. coli</i> (DH10B)</i></b><br>
<ul>
<ul>
<li>Experiment: <br>  
<li>Experiment: <br>  
-
For the chemical transformation the standard protocol was followed.
+
For the chemical transformation the standard <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a> was followed.
<li>Observations & Results: <br>
<li>Observations & Results: <br>
-
The transformation was successful since all plates showed numeours colonoes except the negative control.However, on the plastes with 18K and 18O few red colonies could be observed. These were again rejected.  
+
The transformation was successful since all plates showed numeours colonies except the negative control. However, on the plastes with 18K and 18O few red colonies could be observed. These were again rejected.  
</ul>
</ul>
<br></td></tr>
<br></td></tr>
</table>
</table>
-
 
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<ul>
<ul>
<li>Experiment: <br>  
<li>Experiment: <br>  
-
Over night cultures were preaped of three colonies of each constructs in order to isolate the plasmids.
+
Over night cultures were prepapred of three colonies of each constructs in order to isolate the plasmids.
<br> </li>
<br> </li>
</ul>
</ul>
Line 618: Line 109:
<li>Experiment:  <br>
<li>Experiment:  <br>
Minipreps were conducted with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.  
Minipreps were conducted with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.  
-
<li>
+
<br>
-
</ul>
+
<br>
<br>
<b>V06_08_2  Test digestion of the new <i>flhDC</i>-promoter constructs</b><br>
<b>V06_08_2  Test digestion of the new <i>flhDC</i>-promoter constructs</b><br>
-
<ul>
 
<li>Experiment:  <br>
<li>Experiment:  <br>
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In order to prove correct insertion of <i>flhDC</i> a test digestion was performed using SpeI and XbaI according to the protocol.<br>  
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In order to prove correct insertion of <i>flhDC</i> a test digestion was performed using <i>SpeI</i> and <i>XbaI</i> according to the <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>.<br>  
<li>Observations & Results: <br>
<li>Observations & Results: <br>
For each construct two to three clones hosting the correctly inserted gene in the plasmid could be obtained. Those colonies still containing the rfp gene and were thus rejected.</li>
For each construct two to three clones hosting the correctly inserted gene in the plasmid could be obtained. Those colonies still containing the rfp gene and were thus rejected.</li>
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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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<a href="https://2012.igem.org/Team:Goettingen/Team">Team</a>;
 
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<a href="https://2012.igem.org/Team:Goettingen/Modeling">Modeling</a>;
 
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<a href="https://2012.igem.org/Team:Goettingen/Notebook">Notebook</a>;
 
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<a href="https://2012.igem.org/Team:Goettingen/Saftey">Saftey</a>;
 
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Latest revision as of 17:17, 22 September 2012

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#2 Speed Improvement - 6th week

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V06_04


Chemical retransformation of the flhDC-promoter constructs into E. coli (DH10B)
  • Experiment:
    For the chemical retransformation the standard protocol for transformation was followed. The following constructs were transformed into E. coli:

    20E - #2
    20E - #3
    18M - #2
    18M - #3
    18C - #1
    18C - #2

  • Observations & Results:
    The retransformation was successful since all plates showed numeours colonies except the negative control.


V06_05


V06_05_1 Preparative double digestion of flhDC and plasmids containing further promoters
  • Experiment:
    In order to clone flhDC behind further promoters, the purified PCR product was digested with XbaI and PstI, whereas the BioBrick plasmids were cut with SpeI and PstI. The digestion was performed as described in the protocoll. This time the follwing promoters were used:

    J23113 - 20G - x21
    J23109 - 2G - x106
    J23114 - 20I - x256
    J23106 - 18O - x1185
    J23104 - 18K - x 1831


V06_05_2 Insertion of flhDC into the BioBrick plamids
  • Experiment:
    The ligation of the digested flhDC with the BioBrick plasmids was conducted as described in the protocol.


V06_06


Chemical transformation of the flhDC-promoter constructs into E. coli (DH10B)
  • Experiment:
    For the chemical transformation the standard protocol was followed.
  • Observations & Results:
    The transformation was successful since all plates showed numeours colonies except the negative control. However, on the plastes with 18K and 18O few red colonies could be observed. These were again rejected.


V06_07


Preparation of over night cultures
  • Experiment:
    Over night cultures were prepapred of three colonies of each constructs in order to isolate the plasmids.


V06_08


V06_08_1 Miniprep of the new flhDC-promoter constructs
  • Experiment:
    Minipreps were conducted with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.

    V06_08_2 Test digestion of the new flhDC-promoter constructs
  • Experiment:
    In order to prove correct insertion of flhDC a test digestion was performed using SpeI and XbaI according to the protocol.
  • Observations & Results:
    For each construct two to three clones hosting the correctly inserted gene in the plasmid could be obtained. Those colonies still containing the rfp gene and were thus rejected.


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