Fastprep lysis matrix e tube

Total DNA from 48 ileal and10 fecal samples, respectively, were extracted for determination of the microbiota composition. From the 10^− 1 diluted homogenates used for the enumeration of bacteria, 800 μl homogenate from each of the samples was transferred to a 2 mL FastPrep© Lysis matrix E tube (MP Biomedical, Solon, OH, USA) and further homogenized on a FastPrep-24 Instrument (MP Biomedical) at 6 m/sec for 40 s, followed by centrifugation for 15 min at 10000G. Subsequently, 400 μl of the supernatant of each sample were processed on a Maxwell© RSC instrument (Promega Corporation, Mannheim, Germany) applying the RSC PureFood GMO kit (Cat. # AS1600) according to manufacturer’s protocol for extraction of DNA.
DNA concentrations were measured on an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany) before 16S rRNA gene amplicon library preparation.

Nucleic acid extractions were conducted according to a modified bead-beating protocol (Urich et al., 2008 (link)). Soil samples were washed with DEPC-PBS (Diethylpyrocarbonate, 0.1%; 1 × phosphate-buffered saline) to remove the RNAlater. Approximately 0.5 g of soil was added to a FastPrep™ Lysis Matrix E tube (MP Biomedicals, Solon, OH, USA). Hexadecyltrimethylammonium bromide (CTAB) extraction buffer containing 5% CTAB (in 0.7 M NaCl, 120 mM potassium phosphate, pH 8.0) and 0.5 mL phenol-chloroform-isoamylalcohol (25:24:1) was added and shaken in a FastPrep Instrument (MP Biomedicals, Solon, OH, USA) at speed 5–6 for 45 s. After bead beating, the samples were extracted with chloroform and precipitated in a PEG 6000/1.6 M NaCl solution. Pellets were washed with 70% ethanol and re-suspended in molecular biology grade water. Nucleic acids were further purified using the CleanAll DNA/RNA Clean-up and Concentration Micro Kit (Norgen Biotek Corp., Ontario, Canada). Total DNA was quantified using SybrGreen (Leininger et al., 2006 (link)).