Team:Goettingen/week5-2


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#2 Speed Improvement - 5th week

Miniprep of the distinct constitutive promoters

Experiment:
Minipreps were done with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.

V05_29_1 Preparative double digestion of flhDC and pUC18

Experiment:
In order to clone flhDC behind the different promoters, the purified PCR product was digested with XbaI and PstI, whereas the BioBrick plasmids were cut with SpeI and PstI. The digestion was performed as described in the protocol. At first only three promoters were chosen:
J23112 - 20E - x1
J23105 - 18M - x623
J23100 - 18 C - x 2547
After the digestion the plasmids and the PCR product were loaded onto an 1% agarose gel and separated. Subsequently, the desired fragments were cut out and purified using peqGOLD Gel Extraction Kit (Peqlab) according to the manual.
Observations & Results:
For all plasmids fragments of a size of 2.1 kb and 0.9 kb were obtained. Since these are the expected fragment sizes after a successful digestion the products can be used for further cloning.

V05_29_2 Insertion of flhDC into the BioBrick plamids

Experiment:
The ligation of the digested flhDC with the BioBrick plasmids was conducted as described in the protocol.

Chemical transformation of competent E. coli (DH10B)

Experiment:
After the heat inactivation of the ligase at 65°C the ligated plasmids were chemically transformed into E. coli (DH10B) according to the standard protocol.
Observations & Results:
The transformation was successful. No colonies have developd on the negative control plate whereas the positive control featured numerous. Also the plates with the desired constructs featured growth, however, on the 18C plate three red colonies could be observed. This, seems to be due to failed restriction or ligation.

Preparation of over night cultures

Experiment:
Over night cultures were preaped of three colonies of each constructs in order to isolate the plasmids.

V06_01_1 Miniprep of the flhDC-promoter constructs

Experiment:
Minipreps were conducted with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.

V06_01_2 Test digestion of the flhDC-promoter constructs

Experiment:
All constructs were digested with XbaI and SpeI conducted as described in the protocol.
Observations & Results:
For each construct two clones hosting the correctly inserted gene in the plasmid could be obtained. The others still contain the rfp gene and were thus rejected.