alias: cx0708_S1D3_3
title: DNA amplicon sequencing of fungal ITS2: soil sample
design: Fungal communities on roots and in soil samples were analysed using 454-sequencing of the ITS2 region of the ribosome-encoding genes. PCR was conducted using the fITS9 primer  (Ihrmark et al., 2012) and the ITS4 primer (White et al., 1990), extended with 8 bp sample identification tags as described in Ihrmark et al. (2012). The three DNA extracts from each soil sample were amplified using unique identification tags. PCR amplification was conducted in a 2720 Thermal Cycler (Life Technologies, Carlsbad, CA, USA) in 25 킠 reactions (20 ng DNA, 300 nM tagged ITS4 and 1000 nM fITS9, 0.025 U/킠 polymerase (DreamTaq Green, Thermo Scientific, Waltham, MA, USA) in buffer, 5 min at 94 캜 and cycles of 30 s at 94 캜; 30 s at 55 캜; 30s at 72 캜; 7 min at 72 캜). The number of cycles used (approx. 25 for root samples and 30 for soil samples) was determined for each sample separately to avoid oversaturation. The PCR products were purified using the AMPure kit (Beckman Coulter, Brea, CA, USA) and the concentration of the purified product was determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). PCR products were mixed in equal molar proportions into a general sample, which was further purified using the GeneJetTM PCR-Purification Kit (Fermentas) before being freeze-dried. Addition of sequencing adaptors (by ligation) and 454-sequencing were performed by LGC Genomics GmbH (Berlin, Germany) on a GL FLX Titanium system (Roche, Basel, Switzerland).
identifiers.namespace: SUB1646137
identifiers.id: cx0708_S1D3_3