Qiaamp dna stool mini kit columns

Repeated Bead-Beating (RBB) combined with column-based purification was used to extract DNA from human and animal fecal samples according to protocol Q (IHMS_SOP 06 V2 - http://www.microbiome-standards.org/index.php?id=253) of the International Human Microbiome Standards consortium [20 (link)]. Bead-beating was done using the FastPrep™ Instrument (MP Biomedicals, Santa Ana (CA), USA) with 0.1 mm zirconium-silica beads (BioSpec Products, Bartlesville (OK), USA) to homogenize feces. DNA was finally purified by adapting to QIAamp DNA Stool Mini kit columns (Qiagen, Hilden, Germany).
Regarding the isolation of metagenomic DNA from processed food, 200 mg of sample was homogenized in 0.75 ml PBS (pH 7.2), and centrifuged for 3 min at 13,000 x g. Metagenomic DNA was subsequently isolated by the QIAGEN DNeasy Power Food Kit according to the manufacturer’s instructions for DNA isolation from solid food. For isolation of microbial DNA from water samples, 100 ml of collected water was filtered through 0.22 μm mixed cellulose esters membrane filters (Sartorius, Göttingen, Germany) to capture bacteria. One quarter of the filters were used for metagenomic DNA extraction using the QIAGEN DNeasy Power Water kit according to the manufacturer’s protocol.
Upon isolation, DNA concentrations were determined using the Quan-iT PicoGreen dsDNA assay (Invitrogen, Carlsbad (CA), USA).

Stool samples from patients and the donor were frozen and stored at −80 °C, and subsequently approximately 1 g of each sample was freeze-dried (LYOVAC GT 2, Leybold Heraeus). Genomic DNA was extracted from lyophilized samples using the method of Yu and Morrison [23 (link)], combining rapid beating in a FastPrep-24 homogenizer (MP Biomedicals) with purification in QIAamp DNA Stool Mini Kit columns (Qiagen). The concentration and purity of extracted nucleic acids were checked using a NanoDrop 2000c UV–VIS spectrophotometer (Thermo Scientific). DNA extracts were stored at −20 °C until their use.

Total bacterial DNA was extracted from 30 mg lyophilised faeces with the Repeated Bead Beating Plus Column Method [47 (link)]. This protocol includes two steps of bead beating, which were done by TissueLyser (Qiagen, Hilden, Germany) and 0.1 mm zirconium-silica beads (Biospec Products, Bartlesville, USA). Using the QIAamp DNA Stool Mini Kit columns (Qiagen, Hilden, Germany) according to the manufacturer’s protocol, the DNA was isolated by sequential precipitations and finally purified. Purity of total DNA was estimated from the optical density at 230, 260 and 280 nm (Infinite 200 M microplate reader, Tecan, Männedorf, Switzerland). A 260/280 value of ~ 1.8 is generally accepted as pure DNA. The 260/230 ratio is also an indicator of contamination. The expected 260/230 values are commonly in the range of 2.0 - 2.2. For the analysed faecal samples the observed 260/280 ratio was 1.82 ± 0.03, and the 260/230 ratio 2.07 ± 0.18.
Quantitative PCR analysis was performed as described above on all faecal DNA extracts using group-specific primers targeting the 16S rRNA gene of Lactobacillus spp., Bifidobacterium spp., Streptococcus spp. and Clostridium Cluster XIVa which are listed in Table 5. Standard curves were generated using serial dilutions of the purified and quantified PCR products.