Abi geneamp 9700 pcr thermocycler

Before rats were sacrificed, fecal samples were collected. Microbial community genomic DNA was extracted from fecal samples using an E.Z.N.A.® soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer’s instructions. The DNA extract was checked using 1% agarose gel electrophoresis. The final concentration and purity of microbial DNA were measured using a NanoDrop 2000 UV–vis spectrophotometer (Thermo Scientific, Wilmington, NC, USA). The hypervariable region V3–V4 of the bacterial 16S rRNA gene was amplified using primer pairs 338F (5′-ACTCCT ACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGT WTCTAAT-3′) using an ABI GeneAmp® 9,700 PCR thermocycler (Applied Biosystems, Foster City, CA, USA). PCR amplification conditions were as follows: denaturation at 95°C for 3 min, 27 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 45 s, single extension at 72°C for 10 min, and a final extension at 72°C for 10 min. An AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) was used for PCR purification. A Quantus™ Fluorometer (Promega, Madison, WI, USA) was used for quantitative testing. Purified amplicons were pooled in equimolar concentrations, and paired-end sequencing was carried out using an Illumina MiSeq PE 300 platform (Illumina, San Diego, CA, USA).

Microbial genomic DNA extraction from soil samples was performed with the E.Z.N.A. Soil DNA Kit (Omega Bio-tek). DNA quality assays were performed on 1% agarose gels. DNA concentration and purity were determined with a NanoDrop 2000 UV-vis spectrophotometer (Thermo Fisher Scientific) for determination. The V3-V4 region of the bacterial 16S rRNA gene was amplified with primers s338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The fungal ITS2 region was amplified with primers ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2R (5′-GCTGCGTTCTTCATCGATGC-3′). PCR was performed using an ABI GeneAmp 9700 PCR thermocycler (Applied Biosystems). The PCR amplification process was as follows: initial denaturation at 95°C for 3 min, denaturation at 95°C for 27 cycles of 30 s, annealing at 55°C for 30 s, extension at 72°C for 45 s, one-time extension at 72°C for 10 min, and termination at 4°C. A total volume of 20 μL of PCR mixture included 4 μL of 5× TransStart FastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of 5 μM (each) forward and reverse primers, 0.4 μL of TransStart FastPfu DNA polymerase, 10 ng of template DNA, and sufficient ddH₂O. PCR Reactions were performed in triplicate. PCR products were electrophoresed on a 2% agarose gel, then purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences) and quantified using a Quantus fluorometer (Promega).

Using the E.Z.N.A.^® Soil DNA Kit (Omega Bio-Tek, Norcross, GA, USA), total microbial genomic DNA was isolated from fecal samples and kept at −20 °C. An ABI GeneAmp^® 9700 PCR thermocycler (Applied Biosystems, CA, USA) was used to amplify the 16S rRNA gene V3 and V4 regions from DNA using the primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The purified amplification products were sequenced on an Illumina MiSeq PE300/NovaSeq PE250 platform (Illumina, San Diego, CA, USA) using the standard procedure of Shanghai Mayo Bio-pharm Technology Co., Ltd. (Shanghai, China). Using the UPARS software, sequences were clustered by OTU based on 97% similarity. The principal coordinate analysis (PCoA) plots, beta diversity, Venn map, hierarchical clustering, and alpha diversity calculations were performed using the gene cloud data analysis platform (https://www.majorbio.com, accessed on 12 January 2023).