<b>V08_02_1 2<sup>nd</sup> round: Analysis of transformation V08_01</b><br>
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<ul>
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<li>Experiment: <br>Plates and liquid culture were checked for successful transformation.</li>
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</ul>
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<ul>
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<li>Observations & Results: <br>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!</li>
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</ul>
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<br>
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<b>V08_02_2 2<sup>nd</sup> round: Test digestion of the ethanol precipitation samples</b><br>
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<ul>
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<li>Experiment: <br>A test digestion with <i>Eco</i>RI and <i>Pst</i>I was performed on the samples from V08_01_1.</li>
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</ul>
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<ul>
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<li>Observations & Results: <br>The corresponding agarose gel did not show any bands! There was no DNA material present in the ethanol precipitated samples.</li>
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</ul>
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<br>
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<b>V08_02_3 2<sup>nd</sup> round: mutagenesis PCR from V07_30 repeated</b><br>
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<ul>
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<li>Experiment: <br>The PCR was performed according to the established protocol from <a href="https://2012.igem.org/Team:Goettingen/week10-3">week 10</a>.</li>
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 1st 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.
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