Difference between revisions of "Team:Goettingen/Notebook/reporterSystems September"

A PCR was performed to fuse the promotors P_yciAB and P_yciC to xylR.

The annealing temperature was set for 54°C and lasted for 1 minute. The elongatin settings were 2 min and 72°C.

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The sample was purified with the DNA purification kit and eluted in 32 μL. Also, the DNA concentration was measured with the Nanodrop device.

1.5 μL of the digested plasmid were put on an agarose gel next to a not digested plasmid sample.

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The reaction was inactivated for 5 min at 80°C. Afterwards 1 μL fast AP was added and the reaction was then incubated for 10 min at 37°C. The sample was purified with the DNA purification kit and eluted in 32 μL water. The framgemts were ligated with pAC7 and a religation was performed along side the ligation reaction.

Competent DH5α cells were transformed with the ligation samples and the cells were plated on LB Amp plates containing x-gal.

Inoculation of 15 ml LB over night cultures of three colonies each that showed a blue coulor.

A plasmid prep was performed and the DNA concentration was measured using the nanodrop.

A test digest was performed.

The whole digestion sample was transferred to an agarosegel.

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The colones 1 and 3 of P_yciAB-xylR and clone 3 of :P_yciC-xylR were send away for sequnecing. Clone 3 of :P_yciAB-xylR came back positiv.

The strain BP270 was made competent. Next, the competent cells were transformed with pAC7 containing P_yciAB-xylR isolated from clone 3. In a second reaction BP270 cells were transformed using only the empty pAC7 vector

6 colonies of each transformation were streaked out on agar plates to gain single colonies.

To test whether the correct integartion of the construct was successfull, the α amylase-activity was determined using starch agar plates. Here we determined that all picked clones of the transformation using only the empty plasmid were correct while only one of the 6 picked clones was correct for the transformation with the whole construct. The correct clone was used for further experiments.