Team:Goettingen/week13-3


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#3 Chemoreceptor Library - 13th Week

2^nd round: Analysis of plates and liquid culture V07_20

Experiment:
14 clones were transferred to a 5 mL liquid culture (LB + CM) and incubated over night at 37 °C, 180 rpm. The liquid culture was centrifuged at 4800 rpm, 4 °C, 10 min. The pellet was stored at -20 °C.

Observations & Results:
Both the liquid culture and the plates exhibited bacterial growth.

V07_25_1 2^nd round: Plasmid preparation of clones 1-14 for subsequent sequencing

Experiment:
Plasmid preparation was performd with peqGOLD Plasmid Miniprep Kit (PeqLab) following the user manual. DNA concentrations were determined via nanodrop for subsequent sequencing.

V07_25_2 2^nd round: Saturated mutagenesis PCR 200 µL (repeat) and clean-up

Experiment:
The PCR was set up following the protocol from week 10. Elution in 2 x 50 µL.

Observations & Results:
The corresponding agarose gel showed bands of the expected sizes. However, we apparently did lose DNA material. Later analysis suggested that we used the wrong Kit (peqGOLD Gel extraction Kit (PeqLab)) for this clean-up.

V07_26_1 2^nd round: Digestion DpnI/BsaI, 100 µL and subsequent clean-up

Experiment:
The digestion was performed following the established protocol from week 10.

Observations & Results:
The corresponding agarose gel showed bands of the expected sizes. However, we apparently did lose DNA material. Later analysis suggested that we used the wrong Kit (peqGOLD Gel extraction Kit (PeqLab)instead of peqGOLD Cycle-pure Kit (PeqLab)) for this clean-up. Continuous bug beginning V07_25!

V07_26_2 2^nd round: Ligation, 200 µL

Experiment:
The ligation was performed following the established protocol from week 10. Incubation over night at 16 °C.

V07_27_1 2^nd round: Ethanol precipitation of ligation V07_26

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

Observations & Results:
The corresponding agarose gel did not show any bands, meaning that we lost our DNA material. We could not determine where we went wrong, the transformation was carried out anyhow.

V07_27_2 2^nd round: Transformation of electrocompetent cells with mutant plasmid mixture

Experiment:
The preparation of electrocompetent cells and subsequent transformation were performed following the established protocol from week 10.

V07_27_3 2^nd round: Sequencing of isolated mutant plasmids V07_25

Experiment:
For the sequencing reactions (clones 1-14) we used primer VF2. Again, we wanted to check whether our method had introduced a bias.