<li>Experiment: <br>The digestion and subsequent clean-up was performed according to the established protocol from <a href="https://2012.igem.org/Team:Goettingen/week10-3">week 10</a>.</li>
Starpoint of the 2nd round of saturated mutagenesis PCR!
Experiment: For the second round of saturated mutagenesis PCR we used the primers Tar6973Fw and Tar6973Rv. Here, aa residues 69 and 73 will be mutated. The PCR protocol is basically the same as for the first round.
Observations and results: The corresponding agarose gel showed a strong band of the expected size (3769 bp).
V07_17
V07_17_1 Test digestion of Miniprep V07_12
Experiment: A test digestion using EcoRI and PstI in a 20 µL batch was performed according to protocol to confirm that the mutant plasmid pSB1C3_TAR_QC_18C has actually been isolated correctly.
Observations and results: The corresponding agarose gel showed two distinctive bands of the expected sizes which led to the conclusion that the plasmid had indeed been isolated!
V07_17_2 2nd round: Saturated mutagenesis PCR, volume 1 mL
Experiment: The PCR was now set up in a volume of 1 mL to generate a high amount of DNA in order to reach the diversity we needed. The reaction was split to 20 50 µL tubes.
V07_19
V07_19_1 2nd round: PCR clean-up
Experiment: The clean-up was performed according to the established protocol from week 10.
Observations and results: The corresponding gel showed bands of the expected sizes for each reaction tube.
V07_19_2 2nd round: Digestion DpnI/BsaI and clean-up
Experiment: The digestion and subsequent clean-up was performed according to the established protocol from week 10.
Observations and results: The corresponding gel showed bands of the expected sizes for each reaction tube.
V07_19_3 2nd round: Ligation
Experiment: The ligation was performed according to the established protocol from week 10. We set up a volume of 2 mL.
V07_20
V07_20_1 2nd round: Ethanol precipitation of ligation V07_19
Experiment: The ethanol precipitaion was performed according to the established protocol from week 10.
V07_20_2 2nd round: Transformation of electrocompetent cells with mutated plasmid mixture
Experiment: The preparation of electrocompetent cells and the subsequent transformation were performed according to the established protocol from week 10.
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