Team:Goettingen/week6-2

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#2 Speed Improvement - 6th week

Chemical retransformation of the flhDC-promoter constructs into E. coli (DH10B)

Experiment:
For the chemical retransformation the standard protocol for transformation was followed. The following constructs were transformed into E. coli:

20E - #2
20E - #3
18M - #2
18M - #3
18C - #1
18C - #2
Observations & Results:
The retransformation was successful since all plates showed numeours colonies except the negative control.

V06_05_1 Preparative double digestion of flhDC and plasmids containing further promoters

V06_05_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 the flhDC-promoter constructs 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. However, on the plastes with 18K and 18O few red colonies could be observed. These were again rejected.

Preparation of over night cultures

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

V06_08_1 Miniprep of the new flhDC-promoter constructs

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

V06_08_2 Test digestion of the new flhDC-promoter constructs
Experiment:
In order to prove correct insertion of flhDC a test digestion was performed using SpeI and XbaI according to the protocol.
Observations & Results:
For each construct two to three clones hosting the correctly inserted gene in the plasmid could be obtained. Those colonies still containing the rfp gene and were thus rejected.