Ar200 digital refractometer

Density-gradient separation of DNA was performed according to Buckley et al. [23 (link)] with the exception of excluding the secondary bis-benzimide CsCl gradient [24 (link)]. In summary, DNA fragments >4 kb were selected using a BluePippin platform (Sage Science, Beverly, MA, USA) and added to gradient buffer (15 mM Tris-HCl, pH 8.0; 15 mM EDTA; 15 mM KCl) containing 1.762 g ml⁻¹ CsCl in 4.7 ml polypropylene tubes. Samples were ultracentrifuged to isopycnic equilibrium at 164 000 rcf for 66 h at 20 °C in a TLA-110 fixed angle rotor (Beckman Coulter, Brea, CA, USA). Following centrifugation, tubes were fractionated from bottom to top in 100 μl increments via displacement by Milli-Q water. Fraction densities were calculated using an AR200 Digital Refractometer (Reichert Technologies, Depew, NY, USA). Fractions were then desalted using an Ampure XP bead clean-up kit (Beckman Coulter) and stored at −80 °C.

DNA was extracted from 0.4 g of soil using the protocol described by Lueders et al. (2004a) and the Precellys24 Instrument (PeqLab, Erlangen, Germany). Quality and quantity of the nucleic acids were checked on a spectrophotometer (Nanodrop; PeqLab) and gel electrophoresis. Isopycnic centrifugation was performed in CsCl gradients containing 5 ml of CsCl stock solution (1.84 g ml⁻¹) and 1 ml of gradient buffer (0.1 M Tris‐HCl, pH 8; 0.1 M KCl; 1 mM EDTA) including 4 μg of DNA at 20°C at 45 000 r.p.m. for 48 h in a VTI 65.2 vertical rotor (Beckman Coulter, Krefeld, Germany). Prior to centrifugation, density of the gradients was checked via AR200 digital refractometer (Reichert Technologies, Munich, Germany) and adjusted to 1.72 g ml⁻¹ (Lueders et al., 2004a). Centrifuged gradients were fractionated from bottom to top into 12 equal fractions using Perfusor compact S (Braun AG, Melsungen, Germany). The density of each fraction was measured with a refractometer. Afterwards, the fractions were purified as described by Lueders et al. (2004a) and nucleic acids were quantified using a PicoGreen assay.