Team:Goettingen/week11-2


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#2 Speed Improvement - 11th week

Purification of fliC (DH10B), fliC (Salmonella), motA, motB and yhjH

Experiment:
The amplicons were separated via application on an 1% agarose gel and subsequently cut out. The DNA was purified out of the gel slices using peqGOLD Gel Extraction Kit (Peqlab) according to the user manual.

V07_10_1 Preparative double digestion of fliC (DH10B), fliC (Salmonella), motA, motB, yhjH and pUC18

Experiment:
In order to clone the new genes into pUC18, all components were digested with EcoRI and XbaI according to the protocol. The restricted plasmid pUC18 was subsequently separated on a gel in order to verify the digestions success and purify the desired fragments. The other digestion products were investigated via preparation of an 1% analytical gel and directly purified using the peqGOLD Gel Extraction Kit (Peqlab) according to the manual as well.
Observations & Results:
The digestion was successful. For all PCR products as well as for the vector band of the expected size could be obtained and the fragments can now be used for further cloning.

V07_10_2 Insertion of fliC (DH10B), fliC (Salmonella), motA, motB and yhjH into pUC18

Experiment:
The ligation of the digested genes with the plasmid pUC18 was conducted as described in the protocol.

Chemical transformation of fliC (DH10B), fliC (Salmonella), motA, motB and yhjH into E. coli (DH10B)

Experiment:
For the chemical transformation the standard protocol was followed.
Observations & Results:
The transformation was successful since all plates showed numeours colonies except the negative control.

Preparation of over night cultures

Experiment:
Over night cultures of fliC (DH10B), fliC (Salmonella), motA, motB and yhjH in pUC18 were prepared in order to isolate the plasmids.

V07_13_1 Miniprep of fliC (DH10B), fliC (Salmonella), motA, motB and yhjH in pUC18

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

V07_13_2 Test digestion of fliC (DH10B), fliC (Salmonella), motA, motB and yhjH in pUC18

Experiment:
All vectors were digested with EcoRI and XbaI according to the protocol. The restriction products were subsequently separated on a gel in order to determine successfully ligated plasmids.
Observations & Results:
Except for fliC (Salmonella) #1 all picked colonies seem to host plasmids containing the aimed insert.