Mono q 5 50 gl

DNA substrates were prepared as described in Harami et al.^{19 (link)}. Equimolar amounts of the applicable oligonucleotides were mixed in a buffer comprising 10 mM Tris-HCl, pH 7.5, and 50 mM NaCl. Samples were boiled and were left to cool down to room temperature overnight. Samples were purified on a Mono Q anion-exchange column (Mono Q 5/50 GL, Cytiva) using a 0.01–1 M NaCl gradient for elution and fractions were analyzed by PAGE. Fractions containing the desired DNA structures were desalted by using an Amicon Ultra centrifuge filter (Millipore). DNA substrates were aliquoted and stored at –80 °C. Oligonucleotide sequences are listed above.

pTXB3 plasmids containing the coding sequence for the given RecQ helicase construct in-frame with downstream intein- and chitin- binding domain tags were transformed into E. coli B ER2566 cells (New England Biolabs). Cells were grown in 2YT media supplemented with ampicillin. Protein expression was induced with 0.2 mM IPTG (Sigma) at OD₆₀₀ = 0.6 and cells were incubated for 16 h at 18 °C with shaking. Cell lysis and purification on chitin column were done as described for truncated BLM constructs. RecQ and RecQ* were further purified on heparin column as described for truncated BLM constructs; the CM column was omitted.
RecQ-dH binds weakly to the heparin column. Therefore, instead of the heparin column, this protein was purified on a 1-ml Mono Q column (Mono Q 5/50 GL, Cytiva) as follows. The eluted fractions from the chitin column were dialyses in MQ buffer (50 mM Tris-HCl, pH 8.5, 0.1 mM EDTA, 10 v/v% glycerol, and 1 mM DTT) plus 20 mM NaCl overnight. The sample was loaded onto the Mono Q column equilibrated with MQ buffer plus 20 mM NaCl. Protein was eluted with a linear NaCl gradient (20 mM–1 M Nacl in MQ buffer, 50 min, 1 ml/min).
Fractions containing the purified protein were pooled and dialyzed against a storage buffer (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 10 v/v% glycerol, and 1 mM DTT) overnight.

Bead-bound His-tagged RasGAP²³² was eluted in a stepwise gradient of 20 mM, 40 mM, 100 mM, 250 mM, and 500 mM imidazole in lysis buffer. Fractions containing RasGAP²³² were pooled and mixed with hexahistidine-tagged Tobacco Etch Virus (TEV), and dialyzed overnight against lysis buffer to proteolyze the hexahistidine tag and remove imidazole. The solution was then added back to nickel beads and rocked for 1 h at 4 °C to remove TEV and uncleaved protein. The flowthrough containing tagless RasGAP²³² was concentrated and buffer exchanged into 20 mM Tris pH 8 to reduce NaCl concentration to 50 mM. Anion-exchange chromatography was then performed using either MonoQ 5/50 GL (Cytiva) or ResourceQ (1 ml, Cytiva) columns and buffers A and B which were 20 mM Tris pH 8 and 20 mM Tris pH 8, 1 M NaCl, respectively. Next, size-exclusion chromatography (SEC) was performed using HiLoad 16/600 Superdex 75 prep grade (Cytiva) with 20 mM Tris pH 8, 250 mM NaCl buffer.

Bead-bound His-tagged RasGAP^ΔN was eluted in a stepwise gradient of 10 mM, 20 mM, 100 mM, 250 mM, and 500 mM imidazole in lysis buffer. Fractions containing RasGAP^ΔN were pooled and dialyzed overnight in buffer containing 20 mM Tris pH 8, 150 mM NaCl to decrease the salt concentration. The next day the RasGAP^ΔN sample was concentrated and buffer exchanged into 20 mM Tris pH 8 to reduce NaCl concentration further to 50 mM. Anion-exchange chromatography was then performed using either MonoQ 5/50 GL (Cytiva) or ResourceQ (1 ml, Cytiva) columns and buffers A and B which were 20 mM Tris pH 8.5 and 20 mM Tris pH 8.5, 1 M NaCl, respectively. Next, SEC was performed using HiLoad 16/600 Superdex 200 prep grade (Cytiva) with 20 mM Tris pH 8, 150 mM NaCl buffer.

Bead-bound His-tagged H-Ras^1-167 was eluted in a stepwise gradient of 20 mM, 40 mM, 100 mM, 250 mM, and 500 mM imidazole in lysis buffer. Fractions containing H-Ras^1-167 were pooled and mixed with hexahistidine-tagged TEV and dialyzed overnight against lysis buffer to proteolyze the hexahistidine tag and remove imidazole. The solution was then added back to nickel beads and rocked for 1 h at 4 °C to remove TEV and uncleaved protein. The flowthrough containing tagless H-Ras^1-167 was concentrated and buffer exchanged into 20 mM Tris pH 8 to reduce NaCl concentration to 50 mM. Anion-exchange chromatography was then performed using either MonoQ 5/50 GL (Cytiva) or ResourceQ (1 ml, Cytiva) columns and buffers A and B which were 20 mM Tris pH 8 and 20 mM Tris pH 8, 1 M NaCl, respectively. Next, SEC was performed using HiLoad 16/600 Superdex 75 prep grade (Cytiva) with 20 mM Tris pH 8, 150 mM NaCl buffer.

An oligo for Y-shape double hairpins DNA, GW132 (see Supplementary Table S1 for the sequence), received from Integrated DNA technologies, Inc. (Coralville, IA), was first purified by 5% denaturing PAGE with 7 M urea at room temperature. The DNA extracted from gel was purified further through Mono Q 5/50 GL (Cytiva, Marlborough, MA) using 10 mM NaOH with NaCl gradient. The eluted DNA was neutralized with 1 M HEPES–NaOH (pH 7.5), dialyzed against ddH₂O and lyophilized. The lyophilized oligo was dissolved in dsDNA Annealing Buffer: 20 mM HEPES–NaOH (pH7.5), 100 mM NaCl and 10 mM MgCl₂ and self-annealed. Finally, the quality of dsDNA, as a Y-shape form (GW132H), was assessed by running 20% native PAGE at 4°C. GW132H is configured to be blocked with two short hairpins at one end (creating a Y-shape at this end) and two short overhangs (1 and 3 nt 3′ and 5′ overhangs, respectively) at the open DNA end.