Hi everyone,

Has anyone had problems with Golden Gate when cloning multiple big inserts? My backbone’s size is 7254 bp and each of my insert is approx. 5000-5700 bp (I have 4 inserts) and the size of the final product is approx. 17.200 bp. For the assembly reaction I used 75 ng of each insert (all my inserts were precloned into a vector) and 75 ng of the backbone. I used SapI (10 U) type II restriction enzyme from NEB, T4 DNA Ligase (2000 U/ul), T4 DNA ligase buffer (10x) and Nuclease-free H2O to 20 ul. Incubated the mix in a thermocycler with the following conditions: 37 degrees for 5 min, 16 degrees for 5 min --x 30, 60 degrees for 5 min and 4 degrees. Then I used 2 ul of the reaction mix and transformed in NEB stable cells, and then plated on plates with Ampicillin and IPTG/X-gal because the backbone has the lacZ cassete, but I had 0 colonies.