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.
V08_03
V08_03_1 2st round: PCR clean-up
Experiment: 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.
Observations & Results: The corresponding gel showed a band of the expected size.
V08_03_2 2st round: DpnI/BsaI digestion
Experiment: The digestion was performed with a total volume of 200 µL according to protocol.
V08_03_3 2st round: Digestion clean-up
Experiment: 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.
Observations & Results: The corresponding gel showed a band of the expected size.
V08_05
2st 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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