Wizard sv gen pcr clean up system

Primer selection, amplicon generation and purification, amplicon library construction and sequencing were performed essentially as described by Hevia et al. [17 (link)]. Briefly, the primer pair Probio_Uni / Probio_Rev [16 (link)] was used to generate amplicon pools of approximately 200 bp length. The integrity of the PCR amplicons was analyzed by electrophoresis prior to their purification using the Wizard SV Gen PCR Clean-Up System (Promega, Madison, WI), and the Agencourt AMPure XP DNA purification beads (Beckman Coulter Genomics GmbH, Bernried, Germany). Libraries for each run were diluted to 3E9 DNA molecules prior to clonal amplification. Emulsion PCR was carried out using the Ion OneTouch^™ 200 Template Kit v2 DL (Life Technologies, Guilford, CA) and sequencing of the amplicon libraries was carried out on 316 chips using the Ion Torrent PGM system and employing the Ion Sequencing 200 kit (Life Technologies). After sequencing, individual sequence reads were filtered by the PGM software to remove low quality and polyclonal sequences. Sequences matching the PGM 3’ adaptor were also automatically trimmed. All PGM quality-approved, trimmed and filtered data were exported as SFF files.

Fecal samples were collected from WT and KO mice (n = 8–11 for each genotype) receiving either a regular (NC) or a high-fat diet (HFD). All samples were placed immediately into sterile plastic tubes and stored at −80 °C until analysis. DNA was extracted from the samples using the FastDNA SPIN Kit for Soil (MP Biomedicals Inc., Solon, OH, USA) according to the manufacturer’s instructions.
PCR amplification was performed using primers targeting from V3 to V4 regions of the 16S rRNA gene with extracted DNA. The PCR conditions used were 5 min at 95 °C, 35 cycles of 30 s at 94 °C, 30 s at 55 °C and 90 s at 72 °C, followed by 10 min at 72 °C. Amplification was carried out by using a Verity Thermocycler (Applied Biosystems, Forster City, CA, USA). The PCR product was confirmed by using 2% agarose gel electrophoresis and visualized under UV light. The amplified products were purified with the Wizard SV Gen PCR Clean-Up System (Promega, WI, USA). Equal concentrations of purified products were pooled together and followed by a further purification step involving the Agencourt AMPure XP DNA purification beads (Beckman Coulter Genomics GmbH, Bernried, Germany) in order to remove primer dimers. The quality and product size were assessed using a DNA 7500 chip. Mixed amplicons were pooled and the sequencing was carried out according to the manufacturer’s instructions.