Total genomic DNA was extracted from each sample using a QIAamp Fast DNA Stool Mini Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions. The quality of the DNA was visually assessed using 1.0% (w/v) agarose gel electrophoresis, and the DNA concentration was determined using a NanoDrop spectrophotometer (Nano Drop Technologies, Wilmington, DE, USA). The V3–V4 region of the 16S rRNA gene was amplified from genomic DNA using polymerase chain reaction (PCR) (95°C for 2 min, followed by 25 cycles at 95°C for 30 s, 55°C for 30 s, 72°C for 30 s, and a final extension at 72°C for 5 min) with the primers 338F (ACTCCTACGGGAGGCAGCA) and 806R (GGACTACHVGGGTWTCTAAT). The PCR reactions were performed in a triplicate 25 μL reaction system containing 2 μL DNA template, 2.5 μL 10× TranStart Taq buffer, 1 μL forward and reverse primer respectively, 2 μL dNTPs (2.5 mM), 0.25 μL TransStart Taq DNA polymerase, and 16.25 μL ddH2O. The PCR products were purified with a DNA Purification Kit (TIANGEN, Beijing, China), and barcoded V3–V4 amplicons were sequenced using the Illumina HiSeq platform (HiSeq2500 PE250).
Hiseq platform hiseq2500 pe250
Manufactured by Illumina
Sourced in China
The HiSeq 2500 PE250 is a high-throughput DNA sequencing platform developed by Illumina. It is capable of generating up to 800 million reads per run, with read lengths of up to 250 base pairs in paired-end mode. The platform utilizes Illumina's proprietary sequencing-by-synthesis technology to perform massively parallel DNA sequencing.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using hiseq platform hiseq2500 pe250
1
Gut Microbiome Profiling via 16S rRNA Sequencing
2
16S rRNA Gut Microbiome Profiling
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Gut contents were thawed in ice and then were used to extract DNA by using the QIAamp DNA Stool Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The highly variable V4-V5 region of the 16S rRNA gene was selected and amplified with bacterial-specific universal primers 515F (5′-GTGCCAGCMGCCGCGG-3′) and 907R (5′-CCGTCAATTCMTTTRAGT-3′) (Caporaso et al., 2012 (link)). PCR was performed with conditions as follows: initial denaturation at 95°C for 5 min, 35 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 45 s; and extension at 72°C for 10 min. The products of PCR amplification were sent to Mingke Biotechnology Co., Ltd. (Hangzhou, China) for high-throughput sequencing on the Illumina HiSeq Platform (Hiseq2500 PE250).
3
Gut Microbiome Profiling via 16S rRNA Sequencing
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Gastrointestinal samples were thawed on ice, and microbial genomic DNA was extracted using a QIAamp Fast DNA Stool Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's protocol. The integrity of DNA was visually assessed using 1.0% agarose gel electrophoresis and quantified using a Qubit and NanoDrop. The highly variable V4 region of the 16S rRNA gene was amplified from community genomic DNA using the bacterial‐specific universal primers 515F (GTGCCAGCMGCCGCGGTAA) and 806R (GGACTACHVGGGTWTCTAAT). PCR was performed in triplicate using a 25 μl reaction containing 2 μl DNA template, 2.5 μl 10× TransStart Taq buffer, 1 μl each of forward and reverse primers, 2 μl dNTPs (2.5 mM), 0.25 μl TransStart Taq DNA Polymerase, and 16.25 μl ddH2O. The PCR amplification conditions were as follows: initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 53°C for 30 s and elongation at 72°C for 30 s, and finally, a final extension at 72°C for 10 min. PCR products were purified with a Universal DNA Purification Kit (TIANGEN), and barcoded V4 amplicons were sequenced using the Illumina HiSeq platform (HiSeq2500 PE250).
4
Microbial DNA Extraction and 16S rRNA Sequencing
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Each sample was thawed on ice, and microbial genomic DNA was extracted using a QIAamp Fast DNA Stool Mini Kit (QIAGEN, Germany), according to the manufacturer’s protocol. The negative controls (blank control, no adding the gut or stomach sample) were used during the DNA extraction. The integrity of the DNA was visually assessed using 1.0% agarose gel electrophoresis and quantified using a Qubit and NanoDrop. The 16S rRNA gene V4 region was amplified from the extracted DNA using the universal primers 515F (GTGCCAGCMGCCGCGGTAA) and 806R (GGACTACHVGGGTWTCTAAT). The PCR was performed in triplicate using a 25 μL reaction containing ∼40 ng of DNA template, 2.5 μL of 10x TransStart Taq buffer, 1 μL of each forward and reverse primer, 2 μL of dNTPs (2.5 mM), 0.25 μL of TransStart Taq DNA Polymerase, and 16.25 μL of ddH2O. The polymerase chain reaction thermocycling conditions were: 95°C for 5 min, 35 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 45 s, with a final extension step at 72°C for 10 min. All the PCR products from the blank controls (blank extraction control and blank PCR control) were blank in the agarose gel. The products were purified with a DNA Purification Kit (TIANGEN, Beijing), and barcoded V4 amplicons were sequenced using the Illumina HiSeq platform (HiSeq2500 PE250).
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