The Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, United States) was used to control the quality of the DNA, which was subsequently used to perform amplification of the V3–V4 hypervariable regions of prokaryotic16S rDNA. Pairing primers were designed by GENEWIZ (South Plainfield, NJ, United States). The sequence of the forward primer was “CCTACGGRRBGCASCAGKVRVGAAT,” and the reverse primer contained the sequence “GGACTACNVGGGTW TCTAATCC.” Each polymerase chain reaction volume was 25 μL, containing 2.5 μL of TransStart Buffer (TransGen, Beijing, China), 2 μL of dNTPs, 1 μL of each primer, and 20–30 ng of template DNA. Then, the indexed adapters were attached to the ends of the amplicons to generate indexed libraries for subsequent next-generation sequencing using the Illumina platform (San Diego, CA, United States). The libraries were validated using the Qubit3.0 Fluorometer (Thermo Fisher Scientific) and quantified to 10 nmol. The Illumina MiSeq instrument was used to load and sequence the DNA libraries according to the manufacturer’s instructions. Sequencing was performed under the paired-end 250-bp mode. The raw reads were trimmed using the Cutadapt software to generate clean reads.
Transstart buffer
Manufactured by Transgene
Sourced in China
TransStart Buffer is a laboratory reagent that provides a consistent and optimized environment for various molecular biology applications. It is designed to facilitate efficient and reliable DNA or RNA amplification, enzymatic reactions, and other genetic manipulations. The buffer composition is formulated to maintain the stability and activity of the biomolecules involved in these processes.
Automatically generated - may contain errors
2 protocols using transstart buffer
1
16S rDNA Amplification and Sequencing
2
Microbial DNA Extraction and Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Total bacterial genomic DNA was extracted from the whole NGCA specimen using the DNeasy® PowerSoil® Pro Kit (QIAGEN Co., Ltd., Hilden, Germany) according to the manufacturer's instructions. Genomic DNA integrity was verified by agarose gel electrophoresis. The concentration of extracted DNA was quantified using at DS-11 Series spectrophotometer (DeNovix Inc., Wilmington, USA). DNA extracts were placed on dry ice and sent to GENEWIZ Biological Technology Co., Ltd. (Suzhou, China) for sequencing. The purified genomic DNA was used as the template for amplification of the partial 16S rRNA gene using the universal bacterial primers PCR 319F (5′-CCTACGGGNGGCWGCAG-3′) and 806R (5′-GACTACHVGGGTATCTAATCC-3′) to capture the V3–V4 hypervariable regions. The total volume of each polymerase chain reaction was 25 μL, containing 2.5 μL of TransStart Buffer (TransGen, Beijing, China), 2 μL of dNTPs, 1 μL of each primer, and 20–30 ng of template DNA [25 (link)]. With the Index PCR product, the final libraries were purified using AMPure XP beads (Beckman Coulter, Indianapolis, USA) before quantification. Sequencing was performed on an Illumina MiSeq platform (Illumina Inc., San Diego, CA, USA) to generate a 2 × 300 bp paired-end sequence.
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