Team:Goettingen/week11-3

From 2012.igem.org

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</ul>
<ul>
<ul>
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<li>Observations and results: <br>The corresponding gel showed a band of the expected size.</li>
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<li>Observations & Results: <br>The corresponding gel showed a band of the expected size.</li>
</ul><br>
</ul><br>
<b>V07_10_2 1<sup>st</sup> round: Ligation</b><br>
<b>V07_10_2 1<sup>st</sup> round: Ligation</b><br>
<ul>
<ul>
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<li>Experiment: <br>The ligation and subsequent clean-up were carried out according to the protocol of <a href="https://2012.igem.org/Team:Goettingen/week10-3">week 10</a>.</li>
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<li>Experiment: <br>The ligation was carried out according to the protocol of <a href="https://2012.igem.org/Team:Goettingen/week10-3">week 10</a> and incubated over night at 16 °C.</li>
</ul>
</ul>
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<br>
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</td></tr>
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</table>
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<br>
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<table cellpadding="20 px" border="1" bordercolor="black" valign="top">
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<tr bordercolor="black" valign="top">
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<td width="900" bordercolor="black" valign="top">
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<h2><b>V07_11 </b></h2><br>
 +
<b>V07_11_1 1<sup>st</sup> round: Ethanol precipitation of ligated mutated plasmids</b><br>
<ul>
<ul>
-
<li>Observations and results: <br>The corresponding gel showed a band of the expected size.</li>
+
<li>Experiment: <br>The ethanol precipitation was carried out according to <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>, this time with the right volumes! </li>
</ul>
</ul>
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<br></td></tr>
+
<ul>
 +
<li>Observations & Results: <br>The corresponding gel showed a band of the expected size.</li>
 +
</ul><br>
 +
<b>V07_11_2 1<sup>st</sup> round: Transforamtion of electrocompetent cells with the mutant plasmid mixture</b><br>
 +
<ul>
 +
<li>Experiment: <br>Electrocompetent cells were prepared according to <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>. We transformed 200 µL of cells with 10 µL of DNA according to the protocol of <a href="https://2012.igem.org/Team:Goettingen/week10-3">week 10</a>. Dilution plates and liquid culture was incubated over night at 37 °C, 180 rpm.</li>
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</ul>
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<br>
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</td></tr>
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</table>
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<br>
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<table cellpadding="20 px" border="1" bordercolor="black" valign="top">
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<tr bordercolor="black" valign="top">
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<td width="900" bordercolor="black" valign="top">
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<h2><b>V07_12 </b></h2><br>
 +
<b>V07_12_1 1<sup>st</sup> round: Analysis of transformation V07_11 </b><br>
 +
<ul>
 +
<li>Experiment: <br>Both the liquid culture and the dilution plates were checked for bacterial growth. 5 mL LB liquid cultures + CM were prepared for each clone on the plates for subsequent sequencing of the plasmids. The cultures were incubated over night at 37 °C and 180 rpm.</li>
 +
</ul>
 +
<ul>
 +
<li>Observations & Results: <br>Both the liquid culture and the dilution plates were positive for bacterial growth. Plates showed the following results:<br>
 +
10<sup>4</sup> 10 clones<br>
 +
10<sup>5</sup> 2 clones<br>
 +
10<sup>6</sup> 0 clones<br>
 +
</li>
 +
</ul><br>
 +
<b>V07_12_2 1<sup>st</sup> round: Miniprep of liquid culture V07_11</b><br>
 +
<ul>
 +
<li>Experiment: <br>We used 4 x 5 mL liquid culture split to 4 columns for plasmid preparation with the peqGOLD Plasmid Miniprep Kit (PeqLab).
 +
</li>
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</ul>
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<br>
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</td></tr>
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</table>
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<br>
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<table cellpadding="20 px" border="1" bordercolor="black" valign="top">
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<tr bordercolor="black" valign="top">
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<td width="900" bordercolor="black" valign="top">
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<h2><b>V07_13 </b></h2><br>
 +
<b>V07_13_1 1<sup>st</sup> round: Miniprep of clones 1-12 V07_12 </b><br>
 +
<ul>
 +
<li>Experiment: <br>Plasmids from the 1<sup>st</sup> mutation round were prepped using the peqGOLD Plasmid Miniprep Kit (PeqLab) following the user manual. DNA concentrations were determined for subsequent sequencing.</li>
 +
</ul>
 +
<ul>
 +
<li>Observations & Results: <br>Concentrations varied between 111 and 165 ng/µL.
 +
</li>
 +
</ul><br>
 +
<b>V07_13_2 1<sup>st</sup> round: Sequencing</b><br>
 +
<ul>
 +
<li>Experiment: <br> All 12 clones were sequenced using the primers VR, VF2 and two primers that bind inside <i>tar</i> labeled Seq01_TAR and Seq02_TAR. Here we aimed to examine whether a bias towards certain mutations was introduced.<br>
 +
Additionally we sequenced the generated mutagenesis library with primer VF2 in order to visualize the introduction of multiple mutated bases at the three predetermined sites.
 +
</li>
 +
</ul>
 +
<ul>
 +
<li>Observations & Results: <br>We were able to confirm that our mutagenesis technique did not introduce a bias towards certain mutations in the 1<sup>st</sup> mutation round.<br>
 +
The sequencing chromatogram showed multiple overlaying bases at the predictged sites!
 +
</li>
 +
</ul>
 +
<br>
 +
</td></tr>
</table>
</table>
<br>
<br>
-
 
<a href="https://2012.igem.org/Team:Goettingen/Notebook">Back to overview</a><br>
<a href="https://2012.igem.org/Team:Goettingen/Notebook">Back to overview</a><br>
<br>
<br>

Latest revision as of 13:19, 25 September 2012

Deutsch  / English 

#3 Chemoreceptor Library - 11th Week

Back to overview

V07_10


V07_10_1 1st round: Digestion DpnI/BsaI and clean-up
  • Experiment:
    The digestion and subsequent clean-up were carried out according to the protocol of week 10.
  • Observations & Results:
    The corresponding gel showed a band of the expected size.

V07_10_2 1st round: Ligation
  • Experiment:
    The ligation was carried out according to the protocol of week 10 and incubated over night at 16 °C.


V07_11


V07_11_1 1st round: Ethanol precipitation of ligated mutated plasmids
  • Experiment:
    The ethanol precipitation was carried out according to protocol, this time with the right volumes!
  • Observations & Results:
    The corresponding gel showed a band of the expected size.

V07_11_2 1st round: Transforamtion of electrocompetent cells with the mutant plasmid mixture
  • Experiment:
    Electrocompetent cells were prepared according to protocol. We transformed 200 µL of cells with 10 µL of DNA according to the protocol of week 10. Dilution plates and liquid culture was incubated over night at 37 °C, 180 rpm.


V07_12


V07_12_1 1st round: Analysis of transformation V07_11
  • Experiment:
    Both the liquid culture and the dilution plates were checked for bacterial growth. 5 mL LB liquid cultures + CM were prepared for each clone on the plates for subsequent sequencing of the plasmids. The cultures were incubated over night at 37 °C and 180 rpm.
  • Observations & Results:
    Both the liquid culture and the dilution plates were positive for bacterial growth. Plates showed the following results:
    104 10 clones
    105 2 clones
    106 0 clones

V07_12_2 1st round: Miniprep of liquid culture V07_11
  • Experiment:
    We used 4 x 5 mL liquid culture split to 4 columns for plasmid preparation with the peqGOLD Plasmid Miniprep Kit (PeqLab).


V07_13


V07_13_1 1st round: Miniprep of clones 1-12 V07_12
  • Experiment:
    Plasmids from the 1st mutation round were prepped using the peqGOLD Plasmid Miniprep Kit (PeqLab) following the user manual. DNA concentrations were determined for subsequent sequencing.
  • Observations & Results:
    Concentrations varied between 111 and 165 ng/µL.

V07_13_2 1st round: Sequencing
  • Experiment:
    All 12 clones were sequenced using the primers VR, VF2 and two primers that bind inside tar labeled Seq01_TAR and Seq02_TAR. Here we aimed to examine whether a bias towards certain mutations was introduced.
    Additionally we sequenced the generated mutagenesis library with primer VF2 in order to visualize the introduction of multiple mutated bases at the three predetermined sites.
  • Observations & Results:
    We were able to confirm that our mutagenesis technique did not introduce a bias towards certain mutations in the 1st mutation round.
    The sequencing chromatogram showed multiple overlaying bases at the predictged sites!


Back to overview

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