Team:Goettingen/week16-3


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#3 Chemoreceptor Library - 16th Week

2^nd round: Mutagenesis PCR, 1000 µL

Experiment:
The PCR was set up in a volume of 1000 µL following the protocol from week 10.

2^nd round: PCR purification

Experiment:
The PCR purification was performed using the peqGOLD Cycle-pure Kit (PeqLab), modified using columns from the peqGOLD Plasmid Miniprep Kit (PeqLab)! Elution in 100 µL EB.

Observations & Results:
We now seemed to have found the mistake that caused problems with the loss of DNA material in the second mutagenesis round! Miscommunication led to using the wrong kit for purification steps. We decided to use the peqGOLD Cycle-pure Kit (PeqLab) instead of the subsequent ethanol precipitation as well, as this seemed to rise fewer problems.

V08_15_1 2^nd round: DpnI/BsaI digest and purification

Experiment:
The digest was performed according to protocol from week 10. The subsequent purification was carried out using the peqGOLD Cycle-pure Kit (PeqLab)!!!

Observations & Results:
The corresponding agarose gel showed band of the expected sizes! At last, there did not seem to be any loss of DNA material.

V08_15_2 2^nd round: Ligation, 2000 µL

Experiment:
The ligation was performed according to the establoshed protocol from week 10. Incubation over night at 16 °C.

V08_16_1 2^nd round: Purification of ligation from V08_15

Experiment:
The purification of the ligation was now done using the peqGOLD Cycle-pure Kit (PeqLab) with columns from peqGOLD Plasmid Miniprep Kit I (PeqLab) instead of an ethanol precipitation. This way we could be sure to gain enough DNA material to go on with our library generation. Elution in 50 µL EB.

Observations & Results: The corresponding agarose gel FINALLY showed a band of the expected size, indicating that we could purify our DNA material successfully.

V08_16_2 2^nd round: Transformation of electrocompetent cells with mutant plasmid mixture

Experiment:
Electrocompetent cells were prepared according to protocol. Transformation was performed acording to the established protocol from week 10.

V08_17_1 2^nd round: Analysis of the transformation plates and liquid cultures

Experiment:
Both the liquid culture and the plates were investigated. The liquid culture was centrifuged at 4 °C, 4800 rpm for 10 min. The cell pellet was stored at -20 °C.

Observations & Results:
Both the liquid culture and the plates exhibited bacterial growth. Plates were as follows:
10⁴ 31 colonies
10⁵ 2 colonies
10⁶ 0 colonies