Team:Goettingen/week17-2

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#2 Speed Improvement - 17th week

Preparative double digestion of pSB1C3

Experiment:
The vector pSB1C3 in its linearized as well as in its circularized form was digested with EcoRI and PstI as described in the protocol. Afterwards, the fragments of the desired size were cut out and frozen in order to extract the DNA from the gel slices the following day.
Observations & Results:
The gel featured bands of the expected sizes when irradiated with UV rays.

Purification of the flhDC-promoter constructs

Experiment:
The restricted vector was purified using peqGOLD Gel Extraction Kit (Peqlab) according to the manual.
Observations & Results:
After the purification the DNA concentration was measured with the NanoDrop according to which our samples did not contain any DNA. This is also right for the purified flhDC-promoter constructs. Since NanoDrops are not very accurate, the samples were additionally loaded on a 1% agarose gel, which also did not show any bands expect for those of the marker. Since we already have had problems to receive proper DNA concentration with our purification kit and both preparative gels showed the expected band, we assume that the DNA got lost during purification.

Chemical transformation of the eight flhDC-promoter constructs into E. coli BL21

Experiment:
In order to receive fresh colonies for the subsequent preparation of glycerol stocks competent E. coli BL21 cells were transformed with the eight flhDC-promoter constructs. The other strains were neglected for BL21 showed the strongest susceptibility to inserted constructs.
Observations & Results:
The transformation was successful since all plates showed numerous colonies except for the negative control.

Preparation of overnight cultures

Experiment:
In order to prepare the glycerol stocks over night cultures of the eight flhDC-promoter constructs in E. coli BL21 were prepared. Additionally, over night cultures of MG1655 were prepared for a subsequent preparation of competent cells.

V08_24_1 Preparation of glycerol stocks of the eight flhDC-promoter constructs in E. coliBL21 and the mutation library

Experiment:
The glycerol stocks were prepared according to the following protocol. Furthermore, glycerol stock of the mutation library constructed by group 3 were prepared.

V08_24_2 Chemical transformation of the tar-promoter constructs into E. coli BL21

Experiment:
Since we aim to investigate the constructs using Real-Time Quantitative Reverse Transcription PCR, competent BL21 cells were chemically transformed with the ligation products according to the standard protocol.
Observations & Results:
The transformation was successful since numerous colonies were obtained except for the negative control.

V08_24_3 Miniprep of 18C-flhDC

Experiment:
In order to gain further plasmid material Minipreps were conducted using the PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.

V08_24_4 Preparation of chemically competent cells of MG1655

Experiment:
The preparation of the competent cells was performed according to the following protocoll.

Preparation of overnight cultures

Experiment:
In order isolate the plasmids for a subsequent retransformation of correct vectors over night cultures of the eight tar-promoter constructs were prepared.