Team:Goettingen/week14-3

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#3 Chemoreceptor Library - 14th Week

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V07_30

V07_30_1 2^nd round: Analysis of the transformation V07_27

Experiment:
The plates and liquid culture were analyzed.

Observations & Results:
Neither the liquid culture nor the plates exhibited bacterial growth.

V07_30_2 2^nd round: Saturated mutagenesis PCR, 1000 µL

Experiment:
The PCR was set up again, seeing as we lost our DNA material during the ethanol precipitation the previous week.

V07_31

V07_31_1 2^nd round: PCR clean-up

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

Observations & Results:
The corresponding agarose gel showed a band of the expected size.

V07_31_2 2^nd round: DpnI/BsaI digestion and purification

Experiment:
The digestion was performed with a total volume of 300 µL according to protocol of week 10. The purification was now again performed using the peqGOLD Cycle-pure Kit (PeqLab). This should stop the loss of DNA material throughout the purification steps!

Observations & Results:
The corresponding agarose gel showed a band of the expected size.

V07_31_3 1^st round: Ligation

Experiment:
The ligation was set up in a 100 µL batch according to protocol. Incubation over night at 16 °C.

V08_01

V08_01_1 2^nd round: Ethanol precipitation if ligation V07_31

Experiment:
The ethanol precipitation was performed according to the established protocol form week 10.

Observations & Results:
The corresponding agarose gel showed a band of the expected size.

V08_01_2 2^nd round: Transformation of electrocompetent cells with mutant plasmid mixture

Experiment:
Electrocompetent cells were prepared according to protocol. The transformation was performed according to protocol of week 10.

V08_02

V08_02_1 2^nd round: Analysis of transformation V08_01

Experiment:
Plates and liquid culture were checked for successful transformation.

Observations & Results:
No bacterial growth on neither plates nor in liquid culture. Again, our DNA material was presumably lost during the ethanol precipitation. Confirmation see V08__02_2!

V08_02_2 2^nd round: Test digestion of the ethanol precipitation samples

Experiment:
A test digestion with EcoRI and PstI was performed on the samples from V08_01_1.

Observations & Results:
The corresponding agarose gel did not show any bands! There was no DNA material present in the ethanol precipitated samples.

V08_02_3 2^nd round: mutagenesis PCR from V07_30 repeated

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

V08_03

V08_03_1 2^st round: PCR clean-up

Experiment:
The clean-up was performed using peqGOLD Cycle-Pure Kit (PeqLab) following the user manual. We modified the protocol by using columns from the peqGOLD Plasmid Miniprep Kit (PeqLab) because these can bind a higher amount of DNA (30 µg/column). Thus, we lose fewer DNA material in the cleaning steps. 50 µL of elution buffer was used.

Observations & Results:
The corresponding gel showed a band of the expected size.

V08_03_2 2^st round: DpnI/BsaI digestion

Experiment:
The digestion was performed with a total volume of 200 µL according to protocol.

V08_03_3 2^st round: Digestion clean-up

Experiment:
The clean-up was performed using peqGOLD Cycle-Pure Kit (PeqLab) modified by using columns from the peqGOLD Plasmid Miniprep Kit (PeqLab) with an EB volume of 100 µL.

Observations & Results:
The corresponding gel showed a band of the expected size.

V08_05

2^st round: Ligation

Experiment:
The ligation was set up in a 2000 µL batch according to protocol. Incubation over night at 16 °C.

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