Team:Goettingen/week13-3

From 2012.igem.org

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<b>2<sup>nd</sup> round: Analysis of plates and liquid culture V07_20</b><br>
<b>2<sup>nd</sup> round: Analysis of plates and liquid culture V07_20</b><br>
<ul>
<ul>
-
<li>Experiment: <br>Both the liquid culture and the plates exhibited bacterial growth. 14 clones were transferred to a 5 mL liquid culture (LB + CM) and incubated over night at 37 °C, 180 rpm. The liquid culture was centrifuged at 4800 rpm, 4 °C, 10 min. The pellet was stored at -20 °C.</li>
+
<li>Experiment: <br>14 clones were transferred to a 5 mL liquid culture (LB + CM) and incubated over night at 37 °C, 180 rpm. The liquid culture was centrifuged at 4800 rpm, 4 °C, 10 min. The pellet was stored at -20 °C.</li>
 +
</ul>
 +
<ul>
 +
<li>Observations & Results: <br>Both the liquid culture and the plates exhibited bacterial growth. </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>V07_25 </b></h2><br>
<h2><b>V07_25 </b></h2><br>
-
<b>V07_25_1 2<sup>nd</sup> round: Plasmid preparation clones 1-14 for subsequent sequencing</b><br>
+
<b>V07_25_1 2<sup>nd</sup> round: Plasmid preparation of clones 1-14 for subsequent sequencing</b><br>
<ul>
<ul>
<li>Experiment: <br>Plasmid preparation was performd with peqGOLD Plasmid Miniprep Kit (PeqLab) following the user manual. DNA concentrations were determined via nanodrop for subsequent sequencing.</li>
<li>Experiment: <br>Plasmid preparation was performd with peqGOLD Plasmid Miniprep Kit (PeqLab) following the user manual. DNA concentrations were determined via nanodrop for subsequent sequencing.</li>
</ul>
</ul>
<br>
<br>
-
<b>V07_25_2 2<sup>nd</sup> round: Saturated mutagenesis PCR 200 µL (repeat)</b><br>
+
<b>V07_25_2 2<sup>nd</sup> round: Saturated mutagenesis PCR 200 µL (repeat) and clean-up</b><br>
<ul>
<ul>
-
<li>Experiment: <br>The PCR was set up following the protocol from week <a href="https://2012.igem.org/Team:Goettingen/week10-3">10</a>. </li>
+
<li>Experiment: <br>The PCR was set up following the protocol from <a href="https://2012.igem.org/Team:Goettingen/week10-3">week 10</a>. Elution in 2 x 50 µL.</li>
</ul>
</ul>
-
<br>
 
<ul>
<ul>
-
<li>Observations and results: <br>The corresponding agarose gel showed bands of the expected sizes. </li>
+
<li>Observations & Results: <br>The corresponding agarose gel showed bands of the expected sizes. However, we apparently did lose DNA material. Later analysis suggested that we used the wrong Kit (peqGOLD Gel extraction Kit (PeqLab)) for this clean-up. </li>
</ul><br>
</ul><br>
</td></tr>
</td></tr>
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<td width="900" bordercolor="black" valign="top">
<td width="900" bordercolor="black" valign="top">
<h2><b>V07_26 </b></h2><br>
<h2><b>V07_26 </b></h2><br>
-
<b>2<sup>nd</sup> round: </b><br>
+
<b>V07_26_1 2<sup>nd</sup> round: Digestion <i>Dpn</i>I/<i>Bsa</i>I, 100 µL and subsequent clean-up</b><br>
<ul>
<ul>
-
<li>Experiment: <br></li>
+
<li>Experiment: <br>The digestion was performed following the established protocol from <a href="https://2012.igem.org/Team:Goettingen/week10-3">week 10</a>.</li>
 +
</ul>
 +
<ul>
 +
<li>Observations & Results: <br>The corresponding agarose gel showed bands of the expected sizes. However, we apparently did lose DNA material. Later analysis suggested that we used the wrong Kit (peqGOLD Gel extraction Kit (PeqLab)instead of peqGOLD Cycle-pure Kit (PeqLab)) for this clean-up. Continuous bug beginning V07_25!</li>
 +
</ul>
 +
<br>
 +
<b>V07_26_2 2<sup>nd</sup> round: Ligation, 200 µL</b><br>
 +
<ul>
 +
<li>Experiment: <br>The ligation was performed following the established protocol from <a href="https://2012.igem.org/Team:Goettingen/week10-3">week 10</a>. Incubation over night at 16 °C.</li>
</ul>
</ul>
<br></td></tr>
<br></td></tr>
 +
</table>
 +
<br>
 +
<table cellpadding="20 px" border="1" bordercolor="black" valign="top">
 +
<tr bordercolor="black" valign="top">
 +
<td width="900" bordercolor="black" valign="top">
 +
<h2><b>V07_27 </b></h2><br>
 +
<b>V07_27_1 2<sup>nd</sup> round: Ethanol precipitation of ligation V07_26</b><br>
 +
<ul>
 +
<li>Experiment: <br>The ethanol precipitaion was performed following the established protocol from <a href="https://2012.igem.org/Team:Goettingen/week10-3">week 10</a>.</li>
 +
</ul>
 +
<ul>
 +
<li>Observations & Results: <br>The corresponding agarose gel did not show any bands, meaning that we lost our DNA material. We could not determine where we went wrong, the transformation was carried out anyhow.</li>
 +
</ul>
 +
<br>
 +
<b>V07_27_2 2<sup>nd</sup> round: Transformation of electrocompetent cells with mutant plasmid mixture</b><br>
 +
<ul>
 +
<li>Experiment: <br>The preparation of electrocompetent cells and subsequent transformation were performed following the established protocol from <a href="https://2012.igem.org/Team:Goettingen/week10-3">week 10</a>.</li>
 +
</ul>
 +
<br>
 +
<b>V07_27_3 2<sup>nd</sup> round: Sequencing of isolated mutant plasmids V07_25</b><br>
 +
<ul>
 +
<li>Experiment: <br>For the sequencing reactions (clones 1-14) we used primer VF2. Again, we wanted to check whether our method had introduced a bias.</li>
 +
</ul>
 +
<ul>
 +
<li>Observations & Results: <br>We could confirm that a bias was not introduced.</li>
 +
</ul><br>
 +
</td></tr>
</table>
</table>
<br>
<br>

Latest revision as of 13:21, 25 September 2012

Deutsch  / English 

#3 Chemoreceptor Library - 13th Week

Back to overview

V07_24


2nd round: Analysis of plates and liquid culture V07_20
  • Experiment:
    14 clones were transferred to a 5 mL liquid culture (LB + CM) and incubated over night at 37 °C, 180 rpm. The liquid culture was centrifuged at 4800 rpm, 4 °C, 10 min. The pellet was stored at -20 °C.
  • Observations & Results:
    Both the liquid culture and the plates exhibited bacterial growth.


V07_25


V07_25_1 2nd round: Plasmid preparation of clones 1-14 for subsequent sequencing
  • Experiment:
    Plasmid preparation was performd with peqGOLD Plasmid Miniprep Kit (PeqLab) following the user manual. DNA concentrations were determined via nanodrop for subsequent sequencing.

V07_25_2 2nd round: Saturated mutagenesis PCR 200 µL (repeat) and clean-up
  • Experiment:
    The PCR was set up following the protocol from week 10. Elution in 2 x 50 µL.
  • Observations & Results:
    The corresponding agarose gel showed bands of the expected sizes. However, we apparently did lose DNA material. Later analysis suggested that we used the wrong Kit (peqGOLD Gel extraction Kit (PeqLab)) for this clean-up.


V07_26


V07_26_1 2nd round: Digestion DpnI/BsaI, 100 µL and subsequent clean-up
  • Experiment:
    The digestion was performed following the established protocol from week 10.
  • Observations & Results:
    The corresponding agarose gel showed bands of the expected sizes. However, we apparently did lose DNA material. Later analysis suggested that we used the wrong Kit (peqGOLD Gel extraction Kit (PeqLab)instead of peqGOLD Cycle-pure Kit (PeqLab)) for this clean-up. Continuous bug beginning V07_25!

V07_26_2 2nd round: Ligation, 200 µL
  • Experiment:
    The ligation was performed following the established protocol from week 10. Incubation over night at 16 °C.


V07_27


V07_27_1 2nd round: Ethanol precipitation of ligation V07_26
  • Experiment:
    The ethanol precipitaion was performed following the established protocol from week 10.
  • Observations & Results:
    The corresponding agarose gel did not show any bands, meaning that we lost our DNA material. We could not determine where we went wrong, the transformation was carried out anyhow.

V07_27_2 2nd round: Transformation of electrocompetent cells with mutant plasmid mixture
  • Experiment:
    The preparation of electrocompetent cells and subsequent transformation were performed following the established protocol from week 10.

V07_27_3 2nd round: Sequencing of isolated mutant plasmids V07_25
  • Experiment:
    For the sequencing reactions (clones 1-14) we used primer VF2. Again, we wanted to check whether our method had introduced a bias.
  • Observations & Results:
    We could confirm that a bias was not introduced.


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