Magabio soil feces genomic dna purification kit

Fresh tail stool samples of more than 0.5 g were collected from all participants with informed consent at hospital, frozen at −80°C within an hour, and stored until use. DNA was extracted from each sample and purified with the MagaBio Soil/Feces Genomic DNA Purification Kit (Hangzhou Bioer Technology Co. Ltd.). The concentration and purity of isolated DNA were detected with Thermo NanoDrop One (Thermo Fisher Scientific, MA, USA). PCR was measured in BioRad S1000, targeting the V4 hypervariable region of the bacterial 16S rRNA gene with the forward primer 341F (5′-CCTAYGGGRBGCASCAG-3′) and the reverse primer 806R (5′-GGACTACNNGGGTATCTAAT-3′). Sequencing library was constructed with NEBNext Ultra II DNA Library Prep Kit for Illumina^® (New England Biolabs, MA, USA) according to the manufacturer's instruction and then assessed on the Qubit@ 2.0 Fluorometer (Thermo Fisher Scientific, MA, USA). Pair-end reads (250 bp × 2) were performed by the Guangdong Magigene Biotechnology Co. Ltd. (Guangzhou, China) on an Illumina Nova6000 platform (Illumina Inc., CA, USA).

Deoxyribonucleic acid extraction and purification were conducted with a MagaBio Soil/Feces Genomic DNA Purification Kit (Bioer, Hangzhou, China) according to the manufacturer’s instruction. The V4 region of the 16S rRNA gene was amplified by PCR with the universal primers 515F (5-GTGCCAGCMGCCGCGGTAA-3) and 806R (5-GGACTACHVGGGTWTCTAAT-3) using a TaKaRa Premix Taq^® Version 2.0 (TaKaRa Biotechnology Co., Dalian, China) on a BioRad S1000PCR instrument (Bio-Rad Laboratory, Hercules, CA, USA). The PCR system was 50 μl in total, and it contained 25 μl 2 × Premix Taq, 1 μl of forward primer, 1 μl of reverse primer, 50 ng of template DNA, and double-distilled water (ddH2O) to make up the total volume. PCR was performed using the following conditions: 5 min of denaturation at 94°C, 30 cycles of denaturation at 94°C for 30 s, annealing at 52°C for 30 s, and elongation at 72°C for 30 s, and a final extension at 72°C for 10 min. The 16S rRNA gene amplicons were purified and used for sequencing library construction according to the manufacturer’s instructions for the NEBNext ^® Ultra™ II DNA Library Prep Kit for Illumina ^® (New England Biolabs, Ipswich, MA, USA). The constructed amplicon library was sequenced using the Illumina Nova 6,000 platform.

After being removed from the sampler, the stool was diluted 5X with molecular grade water and homogenized with a vortex. The stool suspension (250 µl) was used for DNA extraction. The genomic DNA was purified and isolated with MagaBio Soil/Feces Genomic DNA Purification Kit (Hangzhou Bioer Technology, Hangzhou, Zhejiang, China). The extracted DNA was quantified at an A260/A280 nm ratio with the NanoDrop ND-1000 (Thermo Fisher Scientific, Cleveland, OH, USA). Then extraction from each sample was diluted to approximately 5 ng/µl and stored at – 20 °C for 16S rRNA sequencing.
The 12-bp barcoded primers synthesized by Invitrogen (Invitrogen, Carlsbad, CA, USA) were used to amplify the bacterial 16S rRNAV3-V4 fragments. The PCR mixture contained 25 μl reactions Taq (Takara Biotechnology, Dalian, Liaoning, China), 1 μl of each primer (10 mM), and 3 μl DNA (20 ng/μl) template (final volume: 50 μl). The PCR protocol was as follows: 94 °C for 30 s followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 52 °C for 30 s, and extension at 72 °C for 30 s; followed by a final extension at 72 °C for 10 min. The PCR products were subjected to 1% agarose gel electrophoresis and then sequenced on an Illumina Miseq (PE 300) in the MAGIGENE Genomic Institute. The PCR products were mixed according to the Instructions of the GeneTools Analysis Software (Version 4.03.05.0, SynGene).