Team:Goettingen/week12-3


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#3 Chemoreceptor Library - 12th Week

Starpoint of the 2^nd 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. We used the Miniprep V07_12 of the liquid culture from the first mutagenesis round. It contains multiple plasmids that are mutated for the first three aa sites. The PCR protocol is basically the same as for the first round.

Observations & Results:
The corresponding agarose gel showed a strong band of the expected size (3769 bp).

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 & 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 2^nd 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_1 2^nd round: PCR clean-up

Experiment:
The clean-up was performed according to the established protocol from week 10.

Observations & Results:
The corresponding gel showed bands of the expected sizes for each reaction tube.

V07_19_2 2^nd 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 & Results:
The corresponding gel showed bands of the expected sizes for each reaction tube.

V07_19_3 2^nd 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_1 2^nd round: Ethanol precipitation of ligation V07_19

Experiment:
The ethanol precipitaion was performed according to the established protocol from week 10.

V07_20_2 2^nd 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. We adjusted the volume of liquid culture to 250 mL to reach diversity.