Stool DNA Kits were used to extract fecal genomic DNA from the pellets (Omega Bio-tek, Inc., Norcross, GA, USA). We used primers F1 and R2 (5′-CCTACGGGNGGCWGCAG-3′ and 5′-GACTACHVGGGTATCTAATCC-3′) located in 16S rRNA gene 341–805 of Escherichia coli for amplifying the V3–V4 regions of the sample. The sequencing library was constructed and paired-end reads were generated on a MiSeq platform, as the manufacturer's instructions. FASTQ software was used for merging and filtering the sequencing data. Diversity indices were calculated by QIIME2. A network analysis was conducted by bacterial genera. We calculated Spearman rank correlations, and if the Spearman rank correlation coefficient was greater than 0.8 and a significance of P < 0.05, we considered that to be a robust correlation. Gephi software was used to construct correlation networks with robust correlations.
Stool dna kit
Manufactured by Omega Bio-Tek
Sourced in United States
The Stool DNA Kit is a laboratory product designed for the extraction and purification of DNA from stool samples. The kit provides the necessary reagents and protocols to efficiently isolate high-quality genomic DNA from human or animal fecal matter.
Automatically generated - may contain errors
Lab products found in correlation
Miseq platform, by Illumina (13 mentions)
Axyprep dna gel extraction kit, by Corning (8 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (7 mentions)
E z n a, by Omega Bio-Tek (6 mentions)
Nanodrop 2000 uv vis spectrophotometer, by Thermo Fisher Scientific (5 mentions)
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Primerscript pcr kit perfect real time, by Takara Bio (3 mentions)
Sybr premix ex taq reagent, by Takara Bio (3 mentions)
Qiaquick gel extraction kit, by Qiagen (3 mentions)
Quantifluor st, by Promega (2 mentions)
Quantifluor st fluorometer, by Promega (2 mentions)
Gel extraction kit, by Qiagen (2 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Superreal premix probe kit, by Tiangen Biotech (1 mentions)
Transgen ap221 02 transstart fastpfu dna polymerase, by Transgene (1 mentions)
Primer premier 5, by Premier Biosoft (1 mentions)
Sybr qpcr master mix, by Vazyme (1 mentions)
Analysis pipeline version 2, by Illumina (1 mentions)
Hiseq 4000 platform, by Illumina (1 mentions)
71 protocols using stool dna kit
1
Gut Microbiome Profiling via 16S rRNA Sequencing
2
Quantifying Gut Microbial Populations
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The caecal and colonic digesta samples were used to extract bacterial DNA using Stool DNA kits (Omega Bio-tek, Doraville, CA). To quantify the microbial population, the primers and fluorescent oligonucleotide probes (Table S5 ) for total bacteria, Bacillus spp., Lactobacillus spp., Escherichia coli, and Bifidobacterium spp. were obtained according to the previously described methods [27 , 28 (link)]. Quantitative real-time PCR was performed using the CFX96 Real-Time PCR Detection System (Bio-Rad, CA, USA). A 25 μL quantitative fluorescent PCR reaction volume was used for counting the total bacteria; it consisted of 1 μL DNA, 1 μL each of upstream and downstream primers, 12.5 μL SYBR Premix Ex Taq™, and 9.5 μL ddH2O. For counting the other bacteria, a 20 μL PCR reaction volume was used; it consisted of 1 μL DNA, 1 μL each of upstream and downstream primers, 0.3 μL probe, 1 μL probe enhancer solution, 8 μL RealMasterMix, and 7.7 μL ddH2O. The PCR conditions and computing method were consistent with those reported by Qi et al. [27 ].
3
Quantifying Gut Microbiome Composition
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Bacterial DNA was extracted from digesta of ileum, cecum, and colon using Stool DNA Kits (Omega Bio-tek, Doraville, CA, USA). The primers and fluorescent oligonucleotide probes (Additional file 1 : Table S2) for total bacteria, Escherichia coli, Lactobacillus spp, Bifidobacterium spp, and Bacillus spp were obtained from the published papers [53 , 54 (link)], which were synthesized by Invitrogen (Shanghai, China). The PCR conditions, reaction system, and calculation method were referring to Qi, et al. (2011) [53 ]. These special kinds of bacteria can be detected by the CFX96 Real-Time PCR Detection System (Bio-Rad, CA, USA).
4
Quantitative DNA Analysis of Gut Microbiome
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Total DNA was extracted from cecum digesta with the Stool DNA Kits (Omega Bio-Tek, Doraville, CA, USA), executed by real-time quantitative PCR using the CFX96 Real-Time PCR Detection system (Bio-Rad Laboratories, Hercules, CA, USA). The total bacteria application program entailed 95 °C for 25 s, followed by 40 cycles of 95 °C for 5 s and 64.5 °C for 25 s. Lactobacillus, E. coli, Bacillus and Bifidobacterium were tested using the SuperReal PreMix (Probe) kit (Tiangen Biotech Co., Ltd., Beijing, China). All results are included for 95 °C for 15 min, followed by 50 cycles of 95 °C for 3 s, and 53 °C for 25 s. All primers used are showed in Table S3 .
5
Quantitative Analysis of Gut Microbiome
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The ileal, cecal, and colonic digesta were collected, and bacterial DNA was extracted with stool DNA kits (Omega Bio-tek, Doraville, CA, USA). The primers and fluorescent oligonucleotide probes (Table S2, Supplementary Materials ) for total bacteria, Eschericha coli, Lactobacillus spp., Bifidobacterium spp., and Bacillus spp. were obtained according to previously published methods [21 ,22 (link)]. Quantitative real-time PCR was accomplished using the CFX96 Real-Time PCR Detection System (Bio-Rad, CA, USA). The PCR conditions and computing method were in accordance with those reported by Qi et al. (2011) [21 ].
6
Bacterial DNA Quantification from Ileal and Cecal Digesta
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Bacterial DNA in the ileal and cecal digesta was extracted by using the Stool DNA Kit (Omega Bio-tek) according to the manufacturer’s instruction. The microbial real-time quantitative PCR was determined as described previously [34 (link)]. Briefly, the number of total bacteria was analyzed by real-time quantitative PCR using SYBR Premix Ex Taq reagents (TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China) and CFX-96 Real-Time PCR Detection System (Bio-Rad Laboratories, Richmond, CA), and the number of Lactobacillus, E. coli and Bifidobacterium was analyzed by real-time quantitative PCR using PrimerScriptTM PCR kit (Perfect Real Time; TaKaRa Biotechnology (Dalian) Co., Ltd., Dalian, China) and CFX-96 Real-Time PCR Detection System (Bio-Rad Laboratories, Richmond, CA) as previously described (Chen et al., 2013). All primers and probes were purchased by TaKaRa Biotechnology (Dalian) Co., Ltd. (Dalian, China), which was listed in Table 3 . For the quantification of bacteria in the test samples, specific standard curves were generated by constructing standard plasmids as presented by Chen et al. (2013) [34 (link)]. In addition, bacterial copies were transformed (log10) before statistical analysis.
7
16S rDNA Sequencing of Gut Microbiome
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The procedure of 16S rDNA sequencing was described in detail according to a previous study (Liu et al., 2018 (link)). The simplified workflow was presented here. The bacterial DNA in digesta were extracted by using Stool DNA Kit (D4015-01, Omega Bio-tek, Norcross, GA), then amplified with the primers of V3-V4 region in bacterial 16S rDNA. The amplicons were purified and then sequenced via the Illumina MiSeq Platform. After quality control and assembly, the clean sequencing data were used for analysis on the online platform of Majorbio Cloud Platform (www.majorbio.com ). α diversity (Chao index and Shannon index), β diversity (Nonmetric MultiDimensional Scaling (NMDS) and Analysis of similarities) and variation between groups at the phylum and genus levels were analyzed. Core Amplicon Sequence Variants (ASVs ) among different GI locations were visualized by Venn plot and Pie plots. Random Forestry Analysis was used to identify the feature ASVs in each part of GI tract (Zheng et al., 2019 (link)). Spearman correlation analysis was performed between core ASVs and BW, jejunal ASVs and jejunal development parameters (villus height, crypt depth and villus height / crypt depth [V/C ]), cecal ASVs and cecal development parameters (cecal length and relative cecal length).
All raw sequencing data have been deposited in the NCBI Sequence Read Archive under the BioProject PRJNA883760.
All raw sequencing data have been deposited in the NCBI Sequence Read Archive under the BioProject PRJNA883760.
8
DNA Extraction from Stool Samples
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DNA was extracted from the supernatant produced using Sheather’s sugar flotation technique with a commercial kit (E.Z.N.A.® Stool DNA Kit, Omega Biotek Inc., Norcross, GA, USA) following the manufacturer’s protocol. DNA was stored at -20˚C before molecular analysis.
9
Rumen Microbiome DNA Extraction and Sequencing
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Total genomic DNA extraction was carried out on the rumen samples using a stool DNA kit (OMEGA Bio-Tek, Norcross, GA, USA). Specifically, the V3-V4 hypervariable regions of the bacterial 16S rRNA gene were targeted for amplification using the universal primers V338F and V806R. The reaction mixture (20 µL) (TransGen AP221-02: TransStart FastPfu DNA Polymerase, TransGen Biotech, Beijing, China) consisted of 4 µL of 5 × FastPfu Buffer, 2 µL of 2.5 mM dNTPs, 0.4 µL of FastPfu polymerase, 0.8 µL of each primer and 10 ng of template DNA. Triplicate PCRs were performed for each sample. The reaction conditions consisted of an initial denaturing step for 3 min at 95 °C; followed by 27 cycles of 30 s at 95 °C, 30 s at 55 °C, 45 s at 72 °C; and 10 min at 72 °C. Amplification products were run in 2% agarose gels and purified with the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). Purified PCR products were quantified fluorometrically. Equimolar ratios of total products were pooled and sequenced with the Illumina MiSeq platform at Majorbio Bio-Pharm Technology Co., Shanghai, China.
10
Ileal Chyme Bacterial DNA Extraction and Quantification
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The total bacterial DNA in ileal chyme (n = 6 for each treatment) was extracted with the Stool DNA Kit (Omega Bio-Tek Doraville, CA, USA) according to the manufacturer’s manual. Primers and probes (Table S2 ) were designed via Primer Premier 5.0 (Premier Biosoft International, Palo Alto, CA, USA).
Microbial RT-qPCR analysis and measurement was carried out as described by Chen et al. [26 (link)]. The total number of bacteria was determined with CFX-96 RT-qPCR and SYBR®-qPCR Master Mix (Vazyme Biotech Co., Ltd., Nanjing, China) in a reaction volume of 25 μL, while the numbers of Lactobacillus, Bifidobacterium, Bacillus, and E. coli were determined with CFX-96 RT-qPCR with SuperReal PreMix (TIANGEN Biotechnology Co., Ltd., Tianjin, China) in a reaction volume of 20 μL. Microbial data were converted into log10- for analysis referring to the standard curve made by Chen et al. [26 (link)].
Microbial RT-qPCR analysis and measurement was carried out as described by Chen et al. [26 (link)]. The total number of bacteria was determined with CFX-96 RT-qPCR and SYBR®-qPCR Master Mix (Vazyme Biotech Co., Ltd., Nanjing, China) in a reaction volume of 25 μL, while the numbers of Lactobacillus, Bifidobacterium, Bacillus, and E. coli were determined with CFX-96 RT-qPCR with SuperReal PreMix (TIANGEN Biotechnology Co., Ltd., Tianjin, China) in a reaction volume of 20 μL. Microbial data were converted into log10- for analysis referring to the standard curve made by Chen et al. [26 (link)].
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