title: GSM4494572: Toehold_OFF_state_bin_1; synthetic construct; OTHER
library_construction_protocol: Cells in collected bins were diluted 10x into fresh LB media with carb selection and allowed to expand overnight at 30C. The expanded cells were then harvested using a MaxiPrep kit (Qiagen #12162). Both ON and OFF inserts were ligated separately into the ColE1 backbone in front of the GFPmut3b-ASV gene using golden gate cloning, as follows. The linearized plasmid backbone was diluted to 500ng total mass. The ON or OFF insert was added according to a 1:1 molar ratio of insert to plasmid backbone. The inserts and backbone dilutions were prepared into 50uL ligation reaction volumes, containing 5uL NEB buffer 3.1 (#B7203S), 5uL T4 ligase buffer (NEB #B0202S), 1uL BsmBI (NEB #R0729), 0.5uL Dpn1 (NEB #R0176), 1uL T4 ligase (NEB #M0202T), and any remaining volume with nuclease-free water. The 50uL reaction was placed into a thermocycler for 100 cycles of two steps: 16C for 10 min and 37C for 10 min. A final enzyme inactivation step at 65C for 15min was done. The ligation products were precipitated out of their reaction buffers using ethanol precipitation. The 50uL ligation reactions were added to 1.5mL Eppendorf tubes containing 150uL of pure ethanol, 5uL 0.3M sodium acetate (pH 5.2), and 1uL GlycoBlue (Invitrogen #AM9515). Tubes were left on dry ice for 20 min and then immediately placed in a 4C tabletop centrifuge and spun at max RPM for 30 min. Tubes were decanted, and 175uL of 70% ethanol was added to the tube containing the pellet. Tubes were spun at max speed for 5 min. Tubes were then removed from the centrifuge, decanted, and allowed to dry for 15 min. Ligation products were then eluted in 4uL TE buffer. For initial library transformation, 50uL EclonI Supreme (Lucigen #60080) cells were given the full 4uL ligation product elution and electro-transformed.
identifiers.namespace: GEO
identifiers.id: GSM4494572