We extracted DNA from feces using the E.Z.N.A. TM Stool DNA Kit (Omega Bio-Tek, Norcross, GA, USA) according to manufacturer protocols. DNA was amplified via PCR using the primers SE47 (5'-CAGCCAAACCTCCCTCTGC-3') and SE48 (5'-CCCGCTTGGTCTTGTCTGTTGC-3') as described previously (Ennis and Gallagher, 1994) . We amplified the amelogenin segment from tissue and feces. According to this method, a male sample contains 2 bands in the agar gel, while a female sample contains 1 band. If 2 bands were found twice out of 6 runs, the fecal sample was regarded as being produced by a male; otherwise the sample was regarded as belonging to a female. PCR amplification was carried out in a total volume of 15 µL consisting of 1.5 µL DNA extract, 1.25 U Premix Ex Taq, 0.5 µL 20 mg/mL bovine serum albumin (Takara, Otsu, Japan), and 10 µM of each primer (SE47, SE48). The PCR protocol consisted of a denaturing step at 94°C for 5 min, followed by 35 cycles at 95°C for 30 s, 62°C for 30 s, and 72°C for 45 s. Final extension was performed at 72°C for 7 min. PCR products were separated by 2% agarose gel electrophoresis for 33 min and then photographed.
E z n a stool dna kit
Manufactured by Omega Bio-Tek
Sourced in United States, Gabon, China
The E.Z.N.A.® Stool DNA Kit is a laboratory product designed for the extraction and purification of DNA from stool samples. It utilizes a silica-based membrane technology to efficiently recover DNA from a variety of stool sources.
Automatically generated - may contain errors
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Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (10 mentions)
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354 protocols using e z n a stool dna kit
1
Fecal DNA Extraction and Sex Determination
2
Profiling Gut Microbiome Diversity in Sow Fecal Samples
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Bacterial genomic DNA was extracted from frozen sow fecal samples with an E.Z.N.A. TM Stool DNA kit (Omega Bio-Tek, Norcross, GA, United States). Sequencing and data analysis were subsequently performed on the Illumina HiSeq PE250 platform by Novogene Institute (Beijing, China), as previously described in Li et al. (18 (link)). The V3–V4 region of 16S rDNA was amplified with the barcoded V4: 515F-806R primers (5′-GTGCCAGCMGCCGCGGTAA-3′ and 5′-GGACTACHVGGGTWTCTAAT-3′, respectively). All sequencing data are available in the NCBI Sequence Read Archive (SRA) under accession PRJNA_783061 (Illumina sequences). Sequences were clustered into the same operational taxonomic units (OTUs) with a 97% similarity threshold. Chao 1 index, Shannon index, and Simpson index were used to ascertain differences in the alpha diversity, and Wilcoxon rank-sum test was used to detect the statistical differences between the two groups. Bray–Curtis distance matrices were calculated for comparison of taxonomic data in beta diversity, and analysis of similarities (ANOSIM) was used to access differences among the microbial communities. All analyses from clustering to alpha and beta diversity were performed in QIIME (V1.7.0) and displayed in R software (V2.15.3).
3
Quantifying Gut Bacterial Populations
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Bacterial DNA in cecal digesta was extracted using the EZNATM Stool DNA kit (Omega BioTek, Doraville, CA, USA). The abundance of total bacteria and specific strains (Escherichia coli, Lactobacillus, Bifidobacterium, and Bacillus) was assessed by RT—qPCR using SYBR Premix Ex Taq reagents (TaKaRa Biotechnology, Dalian, China) and PrimerScriptTM PCR kit (TaKaRa Biotechnology, Dalian, China), respectively. Standard curves were generated using standard plasmids based on Chen et al.’s work (2013) [23 (link)]. Specific primer sequences and probes for RT—qPCR are provided in Table 2 .
4
Gut Microbiome DNA Extraction
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The collected fecal specimens were centrifuged at 12000 × g at room temperature for 5 min and the supernatant was discarded. 200 mg pellet was weighted from each sample and used for total bacterial DNA extraction with the E.Z.N.A Stool DNA Kit (OMEGA Bio-tek, USA) according the manufacturer’s instructions. The quality of DNA was analyzed using Qubit (Invitrogen, USA) and 1% agarose gel electrophoresis. The detail of DNA library construction was described in Supplementary File 1 . The final DNA library was determined the average insert size using the Agilent 2100 Bioanalyzer (Agilent Technologies, USA) and quantified by ABI StepOnePlus Real-Time PCR system (Applied Biosystems, USA).
5
Microbial DNA Extraction and 16S rRNA Amplification
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The E.Z.N.A. ® Stool DNA Kit (Omega Bio-tek, Norcross, USA) was used to isolate high-quality total microbial DNA from stool samples following the manual. The V4–V5 regions of the bacteria 16S ribosomal RNA gene were amplified by PCR. The forward primer used was 515 F: 5’-barcode-GTG CCA GCM GCC GCG G-3’, where the barcode is an eight-base sequence unique to each sample, and the reverse primer was 907R: 5’-CCG TCA ATT CMT TTR AGT TT-3’ [23 (link)]. PCR reactions were performed in triplicate. Each 20 μL reaction mixture contained 10 ng template DNA, 4 μL 5× FastPfu buffer, 2 μL 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), and 0.4 μL FastPfu Polymerase. Reaction was performed at conditions including an initial step at 95 ° C for 2 min, followed by 25 cycles at 95 ° C for 30 s, 55 ° C for 30 s and 72 ° C for 30 s, and a final extension at 72 ° C for 5 min.
6
Ileum Content DNA Extraction
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Total bacterial DNA was extracted from frozen ileum content using the E.Z.N.A® Stool DNA Kit (Omega Bio-Tek, Norcross, USA), according to the manufacturer's instructions. DNA integrity was assessed visually using 1.0% (w/v) agarose gel (containing ethidium bromide) electrophoresis.
7
Fecal DNA Extraction Protocol
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Each fecal sample was washed with sterile water and centrifuged at 2000× g for 15 min to remove potassium dichromate. The extraction of genomic DNA from individual samples was performed using a commercial E.Z.N.A Stool DNA Kit (Omega Bio-tek Inc., Norcross, GA, USA, http://www.omegabiotek.com/ (accessed on 5 July 2020)), following the manufacturer’s recommended procedures. The genomic DNA was stored at –20 °C until its use in PCR amplification.
8
Gut Microbiome Profiling via 16S rRNA Sequencing
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Fecal samples were freshly collected, and DNA was extracted using the E.Z.N.A. stool DNA kit (Omega Bio-tek D4015-02). The V4 region of the 16S rRNA gene was amplified using universal primers (listed in Table S3 ) and sequenced using an Illumina MiSeq apparatus as previously described.62 (link) Paired-end reads were analyzed and classified into operational taxonomic units (OTUs) at >97% identity level using Mothur v.1.40.5.62 (link),63 (link) Taxonomic assignments were determined using the SILVA 16S rRNA reference file release 13264 (link) and the Ribosomal Database Project (RDP) training set version 16.65 (link)
9
16S rRNA Gene Amplification and Sequencing
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DNA was extracted using the Omega Biotek EZNA stool DNA Kit (Georgia, USA). The final DNA concentration was evaluated using a NanoDrop 2000 UV–vis spectrophotometer (Thermo Scientific, Wilmington, NC, USA), and DNA quality was assessed by 1% agarose gel electrophoresis. The V3-V4 region of the 16S ribosomal RNA gene of the bacteria was amplified using primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) in a polymerase chain reaction (PCR) [26 (link)]. The PCR reaction mixture and thermal cycling conditions were consistent with the existing procedures [19 (link)]. For each sample, PCR was performed three times. The Illumina paired-end library was constructed, and the amplicons were then sequenced using the Illumina Miseq PE 300 platform (San Diego, CA, USA). The raw reads were submitted to the NCBI Sequence Read Archive (SRA) database (Bioproject ID: PRJNA997058). The bioinformatics of these paired-end raw sequences are provided in the supplementary materials .
10
Bacterial 16S rRNA Gene Sequencing of Fecal Microbiota
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Faecal samples of subjects were prepare for microbiota analysis. Faecal DNA was extracted using an E.Z.N.A. stool DNA Kit (Omega Bio-tek, USA) according to the manufacturer’s instructions. The V3-V4 regions of the bacterial 16S rRNA genes were amplified using universal primers 338F 5′-ACTCCTACGGGAGGCAGCA-3 and 806R 5′-GGACTACHVGGGTWTCTAAT-3′. PCR amplification of the 16S rRNA gene was carried out in triplicate as follow: template DNA 10 ng, 2.5 mM d NTPs 2 μl, forward and reverse primer (5 μM) 0.8 μl respectively, TransStart fast Pfu polymerase 0.4 μl, 5 × Fast Pfu Buffer 4 μl, and ddH2O in a final volume of 20 μl. PCR amplification program was as follows: an initial activation step with 95°C for 3 min, followed by 27 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s and a final extension at 72°C for 10 min (Dennis et al., 2013 (link)). The quality of PCR products was quantified using QuantiFluor™-ST system (Promega, USA) according to the standard protocols. Then, purified PCR products were sequenced on an Illumina MiSeq platform (Illumina, USA) at Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China.
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