Monarch genomic dna extraction kit

E. coli DH5α (NEB C2987), NEBTurbo (NEB C2984), Klenow (NEB M0210), BL21(DE3) (Agilent 200131), E. coli JM110 (Yanisch‐Perron et al., 1985 (link)), Ni‐NTA resin (Merck 69670), CentriPure P25 columns (emp Biotech Cat. No. CP‐0108), pyruvate kinase (Sigma P1506), lactate dehydrogenase (Sigma L2500), dAMP (Sigma D6375), dCMP (Sigma D7750), dGMP (Sigma D9500), dTMP (Sigma T7004), dADP (Sigma D600), dCDP (Cayman Chemicals 22982), dGDP (Sigma D2950), dTDP (Sigma T9375), NADH (Acros Organics 271100010), PEP (Cayman Chemicals 19192), ATP (Sgima A7699), ADP (Acros Organics 10143940), dNTP Set (NEB N0446), Monarch genomic DNA extraction kit (NEB T3010), 10 kDa MWCO columns (Merck 10125580), tetrabutylammonium phosphate (Fisher Scientific 10569092), ammonium dihydrogen phosphate (Sigma 101126), SeeBlue Plus2 pre‐stained protein ladder (ThermoFisher LC5925), NSR pre‐stained protein ladder (Newmarket Scientific MG20‐10101), 1 kb DNA ladder (NEB N3232), 1 kb plus DNA ladder (NEB N3200), HindIII‐HF (NEB R3104S), Exonuclease III (NEB M0206), benzonase (Sigma E1014).

E. coli were cultured overnight on UTI chromogenic agar (Sigma) at 37 °C. After purity checks single colonies were subcultured overnight in lysogeny broth (LB) (Miller) (shaking, 37 °C). For the majority of isolates DNA was extracted using Monarch Genomic DNA Extraction kit (NEB), but in some instances a lower quantity and quality yield was obtained. In these instances, we observed atypical precipitates and opted for extraction using phenol/chloroform with cetyltrimethylammonium bromide (CTAB). The extracted DNA was sequenced over four runs of MinION (Oxford Nanopore Technology) using R9.4.1 flow cells. Three runs were prepared using Ligation Protocol (LSK-SQK109) and one run was prepared with the Rapid Barcoding Sequencing kit (SQK-RBK004), with both protocols modified to a one-pot implementation.