Team:Goettingen/week15-3

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(Difference between revisions)
 
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<b>V08_08_1 2<sup>nd</sup> round: PCR purification</b><br>
<b>V08_08_1 2<sup>nd</sup> round: PCR purification</b><br>
<ul>
<ul>
-
<li>Experiment: <br>The purification was carried out with the peqGOLD Cycle-pure Kit (PeqLab) following the user manual. Elution in 100 µL EB.</li>
+
<li>Experiment: <br>The purification was carried out with the peqGOLD Gel extraction Kit (PeqLab) following the user manual. Elution in 100 µL EB.</li>
</ul>
</ul>
<ul>
<ul>
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<b>V08_09_1 2<sup>nd</sup> round: PCR purification of V08_08_2</b><br>
<b>V08_09_1 2<sup>nd</sup> round: PCR purification of V08_08_2</b><br>
<ul>
<ul>
-
<li>Experiment: <br>The purification was performed using the peqGOLD Cycle-pure Kit (PeqLab) following the user manual. Elution in 100 µL EB.</li>
+
<li>Experiment: <br>The purification was performed using the peqGOLD Gel extraction Kit (PeqLab) following the user manual. Elution in 100 µL EB.</li>
</ul>
</ul>
<ul>
<ul>

Latest revision as of 14:50, 26 September 2012

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#3 Chemoreceptor Library - 15th Week

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V08_06


V08_06_1 2nd round: Ethanol precipitation of mutated plasmids
  • Experiment:
    The ethanol precipitation was performed according to protocol.

V08_06_2 2nd round: Transformation of electrocompetent cells with the mutant plasmid mixture
  • Experiment:
    Electrocompetent cells were prepared according to protocol. The transformation was performed according to the established protocol.


V08_07


V08_07_1 2nd round: Analysis of transforamtion V08_06
  • Experiment:
    Plates and liquid culture were investigated.
  • Observations & Results:
    Neither the plates nor the liquid culture exhibited bacterial growth. A test gel of the ethano precipitated ligation revealed that, again, our DNA material was lost during the process.

V08_07_2 2nd round: Mutagenesis PCR was set up again, 1000 µL
  • Experiment:
    PCR according to protocol.


V08_08


V08_08_1 2nd round: PCR purification
  • Experiment:
    The purification was carried out with the peqGOLD Gel extraction Kit (PeqLab) following the user manual. Elution in 100 µL EB.
  • Observations & Results:
    Again, we lost a significant amount of DNA material. The subsequent steps (digestion, ligation, transformation) were not carried through because the diversity could not be reached!!

V08_08_2 2nd round: Mutagenesis PCR was set up again, 1000 µL
  • Experiment:
    PCR according to protocol.


V08_09


V08_09_1 2nd round: PCR purification of V08_08_2
  • Experiment:
    The purification was performed using the peqGOLD Gel extraction Kit (PeqLab) following the user manual. Elution in 100 µL EB.
  • Observations & Results:
    Again, we lost a significant amount of DNA material. The subsequent steps (digestion, ligation, transformation) were not carried through because the diversity could not be reached!! We decided to do a test PCR to see whether our protocol worked for the second mutagenesis round.


V08_10


V08_10 2nd round: Test-PCR
  • Experiment:
    The PCR was performed according to the established protocol in a volume of 16 µL.
  • Observations & Results:
    The corresponding agarose gel showed a band of the expected size. This indicated that our PCR worked well!


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