Golden Gate Assembly of Plasmid Constructs

The 20 μl volume assembly reaction solution contained 50 ng receptor vector (~3 k bp), 150 ng of each donor vector (~3 k bp), 1.3 μl of each double-stranded oligonucleotides, 1 μl BsaI (10 U, New England BioLabs, R0535), 1 μl T4 DNA Ligase (2000 U, New England BioLabs, M0202) and 2 μl 10 × T4 DNA Ligase buffer (New England BioLabs). The reaction was performed according to Golden Gate protocol, incubated in a PCR instrument for 10 cycles of 5 min at 37 °C and 10 min at 16 °C, then heated to 37 °C for 15 min, 50 °C for 5 min and then 80 °C for 5 min. 1 μl 25 mM ATP and 1 μl Plasmid Safe DNase (10 U, Epicenter) were then added and incubated at 37 °C for 1 h. 5 μl reaction solution was transformed to chemically competent E. coli Trans5α or PXIDF (pSB1s-X). After 1 h incubation at 37 °C with 200 rpm agitation, cells were plated on LB agar containing appropriate antibiotics [26 (link), 28 (link)]. In all, the assembly reaction just needs a few hours to complete.