DNA plasmids for AFM and bulk integration assays

Negatively supercoiled circular DNA plasmids (pUC19: 2686 bp, pBR322: 4361 bp, M13mp18 RF I DNA: 7249 bp) were obtained commercially (New England Biolabs, Ipswich, MA, USA). To construct the 1800 bp plasmid, commercial pUC19 plasmid (New England Biolabs, Ipswich, MA, USA) was linearized with two primers: FW pUC19 (EcoRV) (5′-GATATCCGTAAAAAGGCCGCG-3′) and REV pUC19 (BspQI) (5′-GCTCTTCCCTTAGACGTCAGGTGGC-3′). The linearization reaction was carried out via PCR reaction with a Phusion master mix (2× Phusion High-Fidelity PCR Master Mix, Thermo Fisher Scientific, Inc., Waltham, MA, USA). One nanogram DNA was used as template for the PCR reaction. The PCR program was: 98 °C 2 min, 30 × (98 °C 10 s, 55 °C 10 s, 72 °C 36 s), 72 °C 5 min. After the PCR reaction was completed, the product was analyzed on a 1% (w/v) agarose gel. The PCR product was directly used in a ligation reaction supplemented with DpnI and T4 Polynucleotide Kinase (PNK; New England Biolabs, Ipswich, MA, USA). DpnI was used to remove all initial DNA template and T4 PNK was used to phosphorylate the DNA for re-ligation. The ligation reaction (1 µl T4 Ligase, 1 µl ATP (10 mM), 0.5 µl PEG-6000, 1 µl T4 PNK, 1 µl DpnI, and 5.5 µl PCR reaction mixture), buffered in CutSmart buffer, was incubated for 15 min at 37 °C, continued with 22 °C for 45 min. One microliter of the reaction was used to transform E. coli DH5alpha cells (New England Biolabs). Few clones were picked for overnight cultures to isolate their plasmid (QIAprep Spin Miniprep Kit, Qiagen, Hilden, Germany). Sequence analysis of the full plasmid confirmed its sequence.

Topologically relaxed pBR322 was generated from commercially available, negatively supercoiled pBR322, by treatment with Wheat Germ Topoisomerase I (Inspiralis, Ipswich, UK), in the PFV reaction buffer at 37 °C. The reaction product was purified using phenol–chloroform extraction and ethanol precipitation. The pellet was resuspended in Tris-HCl buffer (10 mM, pH 8.0). Positively supercoiled pBR322 was generated starting from topologically relaxed pBR322 (see above) employing the archaeal histone-like protein rHmfb²⁰. Under conditions of low-to-intermediate ionic strength, rHmfb wraps DNA in a right-handed toroidal manner. Topoisomerization and subsequent protein removal results in positively supercoiled plasmid DNA. We followed the protocol for production of rHmfb and its use in the generation of positively supercoiled DNA, as originally reported by LaMarr et al.²⁸. Briefly, relaxed pBR322 (500 ng) and rHmfb (250 ng) were incubated for 20 min at 37 °C, in a 25 μL volume of aqueous buffer (10 mM Tris-HCl pH 8.0; 2 mM K₂HPO₄; 1 mM EDTA; 50 mM NaCl). Next, 2.7 μL containing 3 units of Wheat Germ Topoisomerase I (Inspiralis, Ipswich UK) in buffer (295 mM Tris-HCl, pH 8.0; 4 mM H₂KPO₄; 11 mM EDTA; 50 mM NaCl) was added to the solution. Topoisomerization at 37 °C was performed for 90 min. Phenol–chloroform extraction and ethanol precipitation were used to purify the reaction products.