Methods

Cloning

Agarose Gel Electrophoresis

Gel Extraction

Gibson Assembly

To purify plasmid DNA we used several kits that were provided by our sponsors. For Minipreps we used Peqlab, Roche, qiagen, bioRon, Genetics, Roth. For Midipreps we used Qiagen, Jetstar, Peqlab. Beforehand o/n cultures of transformed E.coli were prepared. No deviances from the manufacturer's guidelines were incorporated by performing the isolation steps.

Ligation

Oligo Annealing

Plasmid Isolation

First step of Gibson Assemblies was the rigidly calculation of molar amounts of the fragments. We afterwards joined the fragments and filled them up to 5 µL. These 5 µL were added to 15 µL ice chilled Gibson-Mastermix (Gibson et al., 2009), containing 5x ISO-Buffer, Taq-Ligase and T5 Exonuclease. Except Phusion Polymerase (Gibson et al., 2009) we used the same amount of Q5 Polymerase for the Mastermix. The samples were immediately heated to 50°C for one hour. Afterwards, the samples were cooled for 3 minutes at room temperature and 3 minutes on ice, before they were transformed into competent E.coli.

Polymerase Chain Reaction (PCR)

In order to amplify different DNA-templates, preferably from plasmids, different PCR approaches were used - with the following component amounts for 50 µl of a total volume. 31.5 µl of water, 10 µl of 5x Q5 Reaction Buffer, 4 µl of 2.5 mM dNTP solution, 1 µl of DNA template (200 ng), 1 µl of 10 µM Forward and Reverse Primer, 1 µl of DMSO and 0.5 µl of Q5 High-Fidelity DNA Polymerase. Apart from Touch-down variants and annealing temperature gradient analyses, thermocycler programs consisted of the guideline annotated below.

Preparative Enzymatic Digest

Transformation