Peg6000

CsCl gradients were prepared as described (Neufeld et al., 2007 (link)). In short, ~2 μg of purified DNA was mixed with 4.8 ml of 7.163 M CsCl aqueous solution and the corresponding volume of gradient buffer (0.1 M Tris, 0.1 M KCl, and 1 mM EDTA) to achieve a final density of 1.725 g/ml. Solution was transferred to 5.1 ml polyallomer tubes (Beckham Coulter, Brea, CA, USA) and centrifuged in an Optima™ L-90K ultracentrifuge fitted with a NVT 90 rotor (Beckham Coulter, Brea, CA, USA) for 40 h at 173,000 g for cellulose gradients, and 66 h at 150,000 g for urea gradients, as longer centrifugation times at lower speeds enhance DNA separation in 15N-labeled gradients (Cadisch et al., 2005 (link)). All samples from the same substrate were set up in the same CsCl batch and run in parallel to minimize potential variations. After centrifugation, 400 μL (cellulose) and 200 μL (urea) fractions were collected drop-wise from the bottom of the tube (total of 12 fractions for cellulose gradients, 24 for urea gradients). The density of each fraction was determined by weighing per triplicate a volume of 100 μL in a fine-scale balance. DNA was precipitated with polyethylene glycol (PEG) 6000 (VWR International, Radnor, PA, USA), washed twice with 70% ethanol and resuspended in sterile double-distilled water.