Pcr cleanup

All plasmids used in this work correspond to single transcriptional units (Level 1 plasmids) that were prepared using Golden Gate assembly from Level 0 parts (T7 Promoter, T7 terminator, Toehold Sensors, Triggers, etc.) (Pollak et al., 2019 (link)). The level 0 parts were previously prepared by Gibson assembly (Gibson et al., 2009 (link)) from PCR linear DNA amplified from gblocks (IDT). All plasmids were commercially sequenced before use. Plasmid DNA input was produced by midi prepping an overnight culture of 200 ml LB with the appropriate strain (Promega, A2492) and cleaned again using PCR cleanup (Promega, A6754). Plasmid DNA inputs were used in a set of concentrations ranging from 0.5 to 10 nM. Input linear DNA was produced by PCR and cleaned with PCR cleanup kit (Promega, A6754), quantified and diluted to desired concentrations using ultra-pure water. A list of all the primers and plasmids used in this work is shown in Supplementary Tables S1, 2, respectively.

³²P-labeled probes were obtained by PCR amplification using a dNTP mix supplemented with [α-³²P]-deoxyadenosine triphosphate and plasmid DNA as template. Labeled probes were column-purified to remove radioactive nucleotides (PCR Clean-up, Promega). For gel shift experiments, ³²P-labeled probes were mixed with different concentration of purified IHF in a binding reaction mixture containing sonicated salmon sperm DNA (10 µg/mL [UltraPure™, Invitrogen]) and Bovine Serum Albumine (200 µg/mL [Fraction V, Sigma Aldrich]) in 40 mM Tris-HCl pH7.5, 50 mM NaCl, 40 mM NH₄Cl, 5 mM MgCl₂, 1 mM CaCl2, 8% glycerol, and 1 mM dithiothreitol (DTT). After incubation during 20 min at room temperature, DNA and DNA complexes were separated on a prerun 5,5% polyacrylamide (acrylamide:bis-acrylamide 29∶1) gel supplemented with 10% triethylene glycol [34] in Tris-Borate buffer. Gels were fixed in 10% trichloroacetic acid for 10 min and exposed to Kodak BioMax MR films.

All primers were ordered from IDT. All PCRs were performed with the KAPA HiFi HotStart ReadyMix (Kapa Biosystems, Roche). All PCR products were purified with Promega Wizard SV-Gel and PCR Clean-Up. All plasmids were derived from the pBEST backbone^{30 (link)} with Ampicillin selection marker and ColE1 origin of replication. Cloning was performed with a E. coli strain (DH5α) and plasmids were extracted with spin column purification (Promega).