Plasmid safe nuclease

For the Golden Gate reaction, initially 250 ng of each of the desired modules were mixed with 100 ng of the destination vector, 2 μl T4 DNA ligase buffer (ThermoFisher Scientific), 1.2 μl T4 DNA ligase (ThermoFisher Scientific) and 1 μl BSAI (New England Biolabs) and 0.2 μl BSA (New England Biolabs) in a total volume of 20 μl. In later experiments, the efficiency of the reaction was increased by combining equimolar amounts of modules (0.05 pmol) with 0.025 pmol of destination vector. The following program was then run on a thermocycler T100 or C1000 (BioRad): 50 cycles of 37 °C for 5 min and 16 °C for 5 min, followed by 37 °C for 30 min, 50 °C for 5 min, and 80 °C for 5 min. After the reaction was finished, 1 μl PlasmidSafe nuclease (Epicentre) and 0.85 μl of 25 mM ATP were added to each reaction, and the reactions were incubated at 37 °C for 60 min followed by 70 °C for 30 min, to remove any intermediate, not fully ligated products. 3 μl of each reaction were then transformed into chemically competent NEB10beta cells (New England Biolabs) and correct clones were selected on kanamycin-containing LB agar plates. Clones were analyzed via PmeI digestions and/or Sanger sequencing to confirm the presence of each individual expression cassette.