Dna ligase

Chemicals were purchased from Sigma-Aldrich (Na₂EDTA·2H₂O, CaCl₂·2H₂O, KH₂PO₄, vitamin B₁₂, ZnCl₂, and spectinomycin sulfate), MP Biomedicals (FeCl₃·6H₂O, CuSO₄·5H₂O, and CoCl₂·6H₂O), Acros (Na₂MoO₄·2H₂O), Amresco (NaOAc, 3M, pH 5.2), Fisher Chemical (MnCl₂·4H₂O and Na₂S₂O₃), and Fisher BioReagents [NaCl, MgSO₄·7H₂O, KCl, NaNO₃, Tris base, H₃BO₃, kanamycin monosulfate, SDS, chloroform, saturated phenol (pH 4.3), and absolute ethanol]. Enzymes, including Q5 DNA polymerase, Taq polymerase, DNA ligase, and restriction enzymes, were purchased from New England Biolabs. DNA isolations and purifications were performed using the Zyppy Plasmid Miniprep kit, DNA Clean & Concentrator, and Zymoclean Gel DNA Recovery kit from Zymo Research. Genomic DNA was isolated using the GenElute Bacterial Genomic DNA kit (Sigma-Aldrich). All other vendors are indicated in the subsequent methods sections.

All chemicals used were reagent grade. Restriction endonucleases, DNA ligase, and DNA polymerase were purchased from NEB (MA, USA). Primers were synthesized by Xcelris (India). All chromogenic substrates and ionic liquids were purchased from Sigma-Aldrich. The active fractions post-purification were pooled and concentrated using 30 kDa cut-off size membranes of Amicon-Ultra-15 (Millipore).

Single-stranded DNA curtains were assembled in microfabricated flowcells according to published protocols (37–40 (link)). Briefly, the template and primer oligonucleotides were annealed by heating to 75°C and cooling at a rate of –1°C min^–1. Annealed circles were ligated with DNA Ligase (NEB, M0202) for 5 h at room temperature. Long ssDNA molecules were generated in 1× phi29 reaction buffer (NEB, M0269S), 500 μM dCTP and dTTP (NEB, N0446S), 0.2 mg ml^–1 BSA(NEB, B9000S), 10 nM annealed circles, and 100 nM phi29 DNA polymerase. The mixture was mixed by pipetting and immediately injected on the flowcell and incubated at 30°C for 20–40 min. All microscope experiments were conducted at 37°C. Images were collected on an inverted Nikon Ti-E microscope in a prism TIRF configuration running NIS Elements (AR 4.30.02). Flowcells were illuminated with 488 and 637 nm lasers (Coherent OBIS) split with a 638 nm dichroic mirror (Chroma). Two-color images were recorded by twin electron-multiplying charge-coupled device (EMCCD) cameras (Andor iXon DU897). Uncompressed TIFF stacks were exported from NIS Elements and further analyzed in FIJI (41 (link)). Data analysis was performed in MatLab R2019a (MathWorks).