Team:Goettingen/week14-3

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<b>V08_03_1 2<sup>st</sup> round: PCR clean-up</b><br>
<b>V08_03_1 2<sup>st</sup> round: PCR clean-up</b><br>
<ul>
<ul>
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<li>Experiment: <br>The clean-up was performed using peqGOLD Cycle-Pure Kit (PeqLab) following the user manual. We modified the protocol by using columns from the peqGOLD Plasmid Miniprep Kit (PeqLab) because these can bind a higher amount of DNA (30 µg/column). Thus, we lose fewer DNA material in the cleaning steps. 50 µL of elution buffer was used.
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<li>Experiment: <br>The clean-up was performed using peqGOLD Gel extraction Kit (PeqLab) following the user manual. 100 µL of elution buffer was used.
</li>
</li>
</ul>
</ul>
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<b>V08_03_3 2<sup>nd</sup> round: Digestion clean-up</b><br>
<b>V08_03_3 2<sup>nd</sup> round: Digestion clean-up</b><br>
<ul>
<ul>
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<li>Experiment: <br>The clean-up was performed using peqGOLD Cycle-Pure Kit (PeqLab) modified by using columns from the peqGOLD Plasmid Miniprep Kit (PeqLab) with an EB volume of 100 µL.
+
<li>Experiment: <br>The clean-up was performed using peqGOLD Gel extraction Kit (PeqLab) with an EB volume of 100 µL.
</li>
</li>
</ul>
</ul>
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b>V07_31_2 2
b>V07_31_2 2
/li>
/li>
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b>V08_03_2 2

Latest revision as of 14:52, 26 September 2012

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#3 Chemoreceptor Library - 14th Week

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V07_30


V07_30_1 2nd round: Analysis of the transformation V07_27
  • Experiment:
    The plates and liquid culture were analyzed.
  • Observations & Results:
    Neither the liquid culture nor the plates exhibited bacterial growth.

V07_30_2 2nd round: Saturated mutagenesis PCR, 1000 µL
  • Experiment:
    The PCR was set up again, seeing as we lost our DNA material during the ethanol precipitation the previous week.


V07_31


V07_31_1 2nd round: PCR clean-up
  • Experiment:
    The PCR clean-up was performed according to the established protocol form week 10.
  • Observations & Results:
    The corresponding agarose gel showed a band of the expected size.

V07_31_2 2nd round: DpnI/BsaI digestion and purification
  • Experiment:
    The digestion was performed with a total volume of 300 µL according to protocol of week 10. The purification was now again performed using the peqGOLD Cycle-pure Kit (PeqLab). This should stop the loss of DNA material throughout the purification steps!
  • Observations & Results:
    The corresponding agarose gel showed a band of the expected size.

V07_31_3 2nd round: Ligation
  • Experiment:
    The ligation was set up in a 100 µL batch according to protocol. Incubation over night at 16 °C.


V08_01


V08_01_1 2nd round: Ethanol precipitation if ligation V07_31
  • Experiment:
    The ethanol precipitation was performed according to the established protocol form week 10.
  • Observations & Results:
    The corresponding agarose gel showed a band of the expected size.

V08_01_2 2nd round: Transformation of electrocompetent cells with mutant plasmid mixture
  • Experiment:
    Electrocompetent cells were prepared according to protocol. The transformation was performed according to protocol of week 10.


V08_02


V08_02_1 2nd round: Analysis of transformation V08_01
  • Experiment:
    Plates and liquid culture were checked for successful transformation.
  • Observations & Results:
    No bacterial growth on neither plates nor in liquid culture. Again, our DNA material was presumably lost during the ethanol precipitation. Confirmation see V08__02_2!

V08_02_2 2nd round: Test digestion of the ethanol precipitation samples
  • Experiment:
    A test digestion with EcoRI and PstI was performed on the samples from V08_01_1.
  • Observations & Results:
    The corresponding agarose gel did not show any bands! There was no DNA material present in the ethanol precipitated samples.

V08_02_3 2nd round: mutagenesis PCR from V07_30 repeated
  • Experiment:
    The PCR was performed according to the established protocol from week 10.


V08_03


V08_03_1 2st round: PCR clean-up
  • Experiment:
    The clean-up was performed using peqGOLD Gel extraction Kit (PeqLab) following the user manual. 100 µL of elution buffer was used.
  • Observations & Results:
    The corresponding gel showed a band of the expected size.

V08_03_2 2nd round: DpnI/BsaI digestion
  • Experiment:
    The digestion was performed with a total volume of 200 µL according to protocol.

V08_03_3 2nd round: Digestion clean-up
  • Experiment:
    The clean-up was performed using peqGOLD Gel extraction Kit (PeqLab) with an EB volume of 100 µL.
  • Observations & Results:
    The corresponding gel showed a band of the expected size.


V08_05


2nd round: Ligation
  • Experiment:
    The ligation was set up in a 2000 µL batch according to protocol. Incubation over night at 16 °C.


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b>V07_31_2 2 b>V07_31_2 2 b>V07_31_2 2 /li> /li> b>V07_31_2 2 /li> b>V08_03_2 2

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