The metagenomic DNA was diluted and used as a template for the V3 + V4 variable region of bacterial 16S rDNA. PCR products were detected by 2% agarose gel electrophoresis and purified by the Axygen®AxyPrep DNA gel extraction kit (Axygen Scientific Inc., Union City, CA, USA). And the quantified by QuantiFluorTM-ST Blue Fluorescence Quantification System (Promega), in which the corresponding proportion was mixed according to the sequencing volume requirement of each sample. A TransStart Fastpfu, DNA Polymerase 20-μL reaction system, was used, including 2.0 μL of 10 × buffer, 2.0 μL of 2.5 m MdNTPs, 0.8 μL of forwarding primer (5 μmol/L), 0.8 μL of reverse primer (5 μmol/L), 0.2 μL of Taq polymerase, 0.2 μL of BSA, 10 μL of template DNA, and 20 μL of ddH2O (23 (link)). The PCR products were then sequenced by the Illumina MiSeq sequencing platform (Illumina, San Diego, CA, USA) by Wuhan Fraser Genetic Information Co., Ltd.
Quantifluor st blue fluorescence quantification system
Manufactured by Promega
Sourced in United States, China
The QuantiFluor™-ST Blue Fluorescence Quantification System is a reagent-based method for quantifying nucleic acids and proteins. It utilizes a fluorescent dye that binds to the target molecules, allowing for sensitive and accurate measurement of their concentrations.
Automatically generated - may contain errors
Lab products found in correlation
Axyprep dna gel recovery kit, by Corning (8 mentions)
Miseq platform, by Illumina (8 mentions)
Axyprep dna gel extraction kit, by Corning (5 mentions)
Miseq sequencing platform, by Illumina (4 mentions)
Abi geneamp 9700, by Thermo Fisher Scientific (2 mentions)
Magnetic soil and stool dna kit, by Tiangen Biotech (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Next ultra dna library prep kit, by New England Biolabs (2 mentions)
Miseq pe300 platform, by Illumina (2 mentions)
Axygen axyprep dna gel extraction kit, by Corning (1 mentions)
E z n a soil dna kit, by Omega Bio-Tek (1 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Ap221 02, by Transgene (1 mentions)
Tianamp stool dna kit, by Tiangen Biotech (1 mentions)
Transstart fastpfu dna polymerase, by Thermo Fisher Scientific (1 mentions)
Geneamp 9700, by Thermo Fisher Scientific (1 mentions)
Tiangen fecal genomic dna extraction kit, by Tiangen Biotech (1 mentions)
Tiangen bacterial genomic dna extraction kit, by Tiangen Biotech (1 mentions)
Nanodrop spectrophotometer nd 3.0 1000, by Thermo Fisher Scientific (1 mentions)
Novaseq pe250, by Illumina (1 mentions)
27 protocols using quantifluor st blue fluorescence quantification system
1
Amplification and Sequencing of Microbial 16S rDNA
2
Gut Microbiome Profiling by 16S rRNA Sequencing
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About 2 g of fresh, naturally eliminated feces were collected from subjects on an empty stomach, loaded into test tubes, and stored at a low temperature of -80 °C.
Genomic DNA was extracted from the subject’s stool, then PCR amplification was performed according to the 338F_806R region, and the PCR products were quantified by QuantiFluorTM-ST Blue Fluorescence Quantification System (Promega, America), mixed in the appropriate ratio according to the requirement of sequencing volume for each sample, followed by Miseq library construction as well as sequencing. The DNA fragments were used as templates and the fixed base sequences on the chip were used as primers for PCR synthesis, and the target DNA fragments to be tested were synthesized on the chip to generate DNA clusters, and then the reaction plate surface was scanned with a laser to read the nucleotide species polymerized up by the first reaction of each template sequence, and the results of the collected fluorescence signals were counted to obtain the sequences of the template DNA fragments.
Genomic DNA was extracted from the subject’s stool, then PCR amplification was performed according to the 338F_806R region, and the PCR products were quantified by QuantiFluorTM-ST Blue Fluorescence Quantification System (Promega, America), mixed in the appropriate ratio according to the requirement of sequencing volume for each sample, followed by Miseq library construction as well as sequencing. The DNA fragments were used as templates and the fixed base sequences on the chip were used as primers for PCR synthesis, and the target DNA fragments to be tested were synthesized on the chip to generate DNA clusters, and then the reaction plate surface was scanned with a laser to read the nucleotide species polymerized up by the first reaction of each template sequence, and the results of the collected fluorescence signals were counted to obtain the sequences of the template DNA fragments.
3
Profiling Cecal Microbiome in Piglets
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Fifteen piglets (five piglets per group) were randomly selected for 16S rRNA MiSeq sequencing. Cecal genomic DNA was extracted from 50 mg of cecal samples using the E.Z.N.A. Soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) following the manufacturer’s protocol. The concentration and purity of extracted bacterial DNA were determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The forward (5′-ACTCCTACGGGAGGCAGCAG-3′) and reverse (5′-GGACTACHVGGGTWTCTAAT-3′) primers were used to amplify the highly variable V3–V4 region of the 16S rRNA gene. PCR was performed using TransGen AP221-02: TransStart FastPfu DNA Polymerase (Illumina, San Diego, CA, USA), with a 20 µL reaction system, and an ABI GeneAmp Model 9700 PCR thermal cycler.
DNA extraction, PCR amplification, and product purification and sequencing were performed with the assistance of Shanghai Meiji Biopharmaceutical Technology Co. Referring to the preliminary quantification results of electrophoresis, the PCR products were detected and quantified using the QuantiFluor™-ST Blue Fluorescence Quantification System (Promega, Beijing, China), and the TruSeqTM DNA Sample Prep Kit was selected for library construction. The purified amplicons were pooled at equimolar levels on the Illumina MiSeq PE300/NovaSeq PE250 platform (Illumina, San Diego, CA, USA) followed by paired-end sequencing.
DNA extraction, PCR amplification, and product purification and sequencing were performed with the assistance of Shanghai Meiji Biopharmaceutical Technology Co. Referring to the preliminary quantification results of electrophoresis, the PCR products were detected and quantified using the QuantiFluor™-ST Blue Fluorescence Quantification System (Promega, Beijing, China), and the TruSeqTM DNA Sample Prep Kit was selected for library construction. The purified amplicons were pooled at equimolar levels on the Illumina MiSeq PE300/NovaSeq PE250 platform (Illumina, San Diego, CA, USA) followed by paired-end sequencing.
4
Intestinal Microbiome Analysis via 16S rRNA Sequencing
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Intestines (n = 4 repeats, 3 intestine samples pooled as one repeat) were used for 16S rRNA gene amplicons sequencing. Briefly, genomic DNA was extracted from 50 mg of tissue samples and detected through 2% agarose gel electrophoresis, amplified using an ABI GeneAmp® 9700 PCR (ABI, Los Angeles, CA, USA), and the primer is 338F-806R. The PCR products were cut and recovered using the AxyPrepDNA gel recovery kit (AXYGEN, New York, USA), subsequently eluted by Tris_HCl [32 (link)]. The PCR products were quantified by the QuantiFluor™-ST Blue Fluorescence Quantification System (Promega, Madison, WI, USA). Miseq amplicon libraries were constructed and sequenced on the Illumina MiSeq-PE25 platform (Illumina, San Diego, CA, USA) in Majorbio Co. (Shanghai, China) [33 (link)]. The data were uploaded to the Majorbio Co., Cloud Platform (https://cloud.majorbio.com ) for result analysis. All 16S rRNA sequence data can be downloaded from the National Center for Biotechnology Information (NCBI) under the project accession PRJNA1043446 (Submission ID: SUB13989547).
5
Illumina-based 16S rRNA profiling
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Specific primers with barcode were synthesized and the V3-V4 region of the 16 S rRNA gene was amplified using TransGen AP221-02. The PCR products were mixed and detected by 2% agarose gel electrophoresis. The products were purified by the AxyPrepDNA Gel Recovery Kit (AXYGEN, USA) and quantified using the QuantiFluor™ -ST Blue Fluorescence Quantification System (Promega, USA). Finally, sequencing was performed by Mejorbio Biopharmaceuticals on the llumina MiSeq platform (Illumina, USA).
UPARSE (version 7.0.1090http://drive5.com/uparse/ ) was used for analysis of OTUs at 97% similarity. Alpha diversity indices (sobs, shannon, simpson, ace, chao, coverage) were calculated by Mothur (version 1.30.2 https://www.mothur.org/wiki/Download_mothur ) to estimate community richness and diversity. The beta diversity of distance matrix was calculated by QIIME (version 1.9.1 http://qiime.org/install/index.html ), the Non-metric multidimensional scaling (NMDS) analysis was performed by Vegan and differences between groups were analysed by Partial Least Squares Discriminant Analysis (PLS-DA). LEfSe analysis was performed to estimate differences of species abundance and the Kruskal-Wallis H test was used to assess the significance of the differences. Correlation coefficients of microbial communities were calculated using Spearman correlation algorithm and visualized by Cytoscape.
UPARSE (version 7.0.1090
6
Gut Microbiome DNA Extraction and Sequencing
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All samples were extracted using the TIANamp Stool DNA Kit (TIANGEN, Sichuan, China). Specific primers with barcodes were synthesized according to the specified sequencing region. PCR amplification was performed according to the manufacturer’s instructions, with three replicates per sample. PCR products from the same samples were mixed and detected by 2% agarose gel electrophoresis. PCR products were recovered using AxyPrepDNA gel recovery kit (AXYGEN Corporation, Silicon Valley, CA, USA). Detection and quantification were performed by QuantiFluor-ST™ Blue Fluorescence Quantification System (Promega, Madison, WI, USA). Purified PCR amplicons were sequenced on the Illumina MiSeq platform at Shanghai Majorbio Bio-pharm Technology Co., Ltd.
7
Fecal Microbiome Analysis by 16S rRNA Sequencing
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16S rRNA gene sequencing was conducted based on the procedures reported previously [32 ]. Briefly, after indicate treatment, the fecal samples of dams were collected under sterile conditions at GD0 and GD21, and the samples of offspring were collected at PD10, PD21 and PD49, respectively. The samples were all stored at −80 °C prior to use. After the total genomic DNA was extracted from samples, the bacterial 16S rRNA was amplified using the forward and reverse primers designed by adding a barcode to primers. The PCR amplification reaction was performed on the ABI GeneAmp 9700 (thermal cyclers from Applied Biosystems, CA, USA) using TransStart Fastpfu DNA polymerase. The PCR products were purified by AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, CA, USA) and quantified by using QuantiFluor™-ST Blue Fluorescence Quantification System (Promega Co., WI, USA). MiSeq library was constructed for preparation of the fragment DNA, and the raw sequencing reads were obtained using Illumina MiSeq platform at Majorbio Bio Tech Co. Ltd (Shanghai, China).
8
Maize Rhizosphere Soil Microbiome Analysis
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The maize rhizosphere soil from normal conditions and medium drought treatment on the 26th day after inoculation were collected and used for high-throughput sequencing. After extracting the DNA of each sample, it was subjected to 1% agarose gel electrophoresis. Specific primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) with barcodes were used to amplify 16S rRNA of the bacterial V3-V4 region. The PCR products were detected and quantified by QuantiFluor™-ST Blue Fluorescence Quantification System (Promega), and then, each sample was mixed in the corresponding proportion. The following thermal program was used for amplification: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 45 s (a total of 30 cycles), and finally, extension at 72°C for 10 min. The reaction products were detected using 2% agarose gel electrophoresis. The Miseq library was constructed with TruSeq™ DNA Sample Prep Kit reagents, and the data were optimized using Trimmomatic and FLASH software after sequencing was completed on the Illumina MiSeq sequencing platform (Majorbio, Shanghai, China).
9
Gut Microbiome DNA Extraction and Sequencing
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Total genome DNA from cecal contents were extracted using Magnetic Soil And Stool DNA Kit (TIANGEN). DNA concentration and purity were monitored on 1% agarosegels. According to the concentration, DNA was diluted to 1 ng/μl using sterile water. PCR amplification of selected V3-V4 variable regions was performed using specific primers with Barcode and high fidelity DNA polymerase according to the selection of sequencing regions. PCR products were examined by 2% agarose gel electrophoresis and the target fragments were recovered by gel cutting using the AxyPrepDNA Gel Recovery Kit (AXYGEN). The PCR amplified recovered products were detected and quantified by reference to the preliminary quantification results of electrophoresis using the QuantiFluor™ -ST Blue Fluorescence Quantification System (Promega), and mixed in the appropriate proportions according to the sequencing volume required for each sample. Libraries were constructed using the NEB Next® Ultra™ DNA Library Prep Kit. Libraries were quality checked by Agilent Bioanalyzer 2100 and Qubit and sequenced after passing the library quality check (Detailed experimental and statistical methods are shown in Additional file 1).
10
Fecal Microbiome Profiling by 16S rRNA Sequencing
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DNA sequencing was performed at Shanghai Lingen Biotechnology Co., Ltd. Fecal microbial DNA was extracted using a fecal DNA kit (Omega Bio-tek, Norcross, GA, USA). The V4–V5 region of the bacterial 16S ribosomal RNA gene was amplified using polymerase chain reaction (PCR) and 515F 5′-barcode-GTGCCAGCMGCCGCGG-3′ and 907R 5′-CCGTCAATTCMTTTRAGTTT-3′ primers. PCR products were quantified using the QuantiFluor™-ST Blue Fluorescence Quantification System (Promega). After Illumina PE250 library construction, sequencing of PCR product libraries was performed on the Illumina MiSeq platform (Illumina Inc., San Diego, CA, USA). The paired-end (PE) reads obtained from sequencing were spliced into a sequence according to the overlapping relationship between them, while the sequence quality was controlled and filtered. Using the QIIME software package (V1.9.0 http://qiime.org/scripts/assign_taxonomy.html ), sequences with similarities ≥97% were clustered into an operational taxonomic unit (OTU) and analyzed taxonomically for species.
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