Team:Goettingen/week15-3


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#3 Chemoreceptor Library - 15th Week

V08_06_1 2^nd round: Ethanol precipitation of mutated plasmids

V08_06_2 2^nd round: Transformation of electrocompetent cells with the mutant plasmid mixture

Experiment:
Electrocompetent cells were prepared according to protocol. The transformation was performed according to the established protocol.

V08_07_1 2^nd round: Analysis of transforamtion V08_06

Observations & Results:
Neither the plates nor the liquid culture exhibited bacterial growth. A test gel of the ethano precipitated ligation revealed that, again, our DNA material was lost during the process.

V08_07_2 2^nd round: Mutagenesis PCR was set up again, 1000 µL

V08_08_1 2^nd round: PCR purification

Experiment:
The purification was carried out with the peqGOLD Gel extraction Kit (PeqLab) following the user manual. Elution in 100 µL EB.

Observations & Results:
Again, we lost a significant amount of DNA material. The subsequent steps (digestion, ligation, transformation) were not carried through because the diversity could not be reached!!

V08_08_2 2^nd round: Mutagenesis PCR was set up again, 1000 µL

V08_09_1 2^nd round: PCR purification of V08_08_2

Experiment:
The purification was performed using the peqGOLD Gel extraction Kit (PeqLab) following the user manual. Elution in 100 µL EB.

Observations & Results:
Again, we lost a significant amount of DNA material. The subsequent steps (digestion, ligation, transformation) were not carried through because the diversity could not be reached!! We decided to do a test PCR to see whether our protocol worked for the second mutagenesis round.

V08_10 2^nd round: Test-PCR

Experiment:
The PCR was performed according to the established protocol in a volume of 16 µL.

Observations & Results:
The corresponding agarose gel showed a band of the expected size. This indicated that our PCR worked well!