2.6. Gateway Cloning

To generate expression vectors, Gateway LR Clonase II Enzyme Mix (Thermo Fisher, 11791) was used. In brief, LR-recombination reactions between Gateway entry vectors containing attL-sites and Gateway destination plasmids containing attR-sites were set up by mixing 150 ng of the entry vector with 150 ng of the destination vector. TE buffer was added to reach a final volume of 8 µL. Then, 2 µL of LR Clonase II Enzyme Mix were added and incubated for 1–3 h at 25 °C. 1 µL of Proteinase K was added to the reaction mix and incubated for 10 min at 37 °C. The reaction mix was transformed into DH5α.