Neb 10 beta competent e coli high efficiency

The vector (Addgene #73037) encoding for copGFP, puromycin and nanoluc was restricted with BamHI and XbaI and used as backbone for a Gibson assembly (New England Biolabs) to insert a gBlock (IDT) encoding for the N-terminus of NoMi (see Figure S1b). The resulting plasmid was restricted with BmgBI and BarI to incorporate the NoMi C-terminus with Gibson assembly and generate the NoMi plasmid.
Each assembly reaction contained approximately 100 ng insert and 50 ng expression vector and was incubated at 50 °C for 30 min - 4 h following the manufacturer’s protocol. The plasmids were transformed into NEB® 10-beta Competent E. coli (High Efficiency) (New England Biolabs) and overnight grown at 37 °C on antibiotic containing agar plates [10 mL Bacto agar (Sigma) with LB medium in a 60 mm dish]. Single colonies were picked, grown in LB broth (Sigma) and extracted using a QIAprep Spin Miniprep Kit (Qiagen). To confirm correct insertion, a restriction digest was performed, and fragments electrophoresed in a 1.5% agarose gel and stained with GelRed (Biotium). Images were acquired under UV light using Azure Biosystems c300 Image. When correct profiles were detected, complete plasmid sequencing using Next-Generation sequencing technology (MGH CCIB DNA Core) was performed to validate plasmid integrity.