Abi geneamp 9700 pcr thermocycler

Each sample, weighing approximately 0.2 g, was subjected to DNA extraction using the QIAamp DNA Stool Mini Kit (Qiagen, Valencia, CA, USA). The extracted DNA samples were stored at −80 °C until further use. For the amplification of the V3–V4 hypervariable region of the bacterial 16S rRNA gene, specific primer pairs 341F (5′–CCTAYGGGRBGCASCAG–3′) and 806R (5′–GGACTACNNGGGTATCTAAT–3′) were used. The PCR amplification was carried out using an ABI GeneAmp^® 9700 PCR thermocycler (ABI, CA, USA). Subsequently, the total DNA extracted from the fish gut samples was sent to Majorbio Bio-Pharm Technology Co., Ltd., located in Shanghai, China, for further analysis using Illumina MiSeq sequencing.

Microbial community genomic DNA was extracted from compost samples using the E.Z.N.A.^® soil DNA Kit (Omega Biotek, Norcross, GA, U.S.) according to the manufacturer’s instructions. The DNA extract was checked on a 2% agarose gel, and the DNA concentration and purity were determined with a NanoDrop 2000 UV‒vis spectrophotometer (Thermo Scientific, Wilmington, USA). The hypervariable region V3-V4 of the bacterial 16S rRNA gene was amplified with primer pairs 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTA AT-3'). The hypervariable region ITS1 of the fungal ITS gene was amplified with the primer pair ITS1F (5'-CTTGGTCATTTAGAGGAAGTAA-3') and ITS2R (5'-GCTG CGTTCTTCATCGATGC-3'). PCR amplification instruments were used with an ABI GeneAmp^® 9700 PCR Thermocycler (ABI, CA, USA).

The V3-V4 variable region of the 16S rRNA gene was amplified by PCR using the primer pairs 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) by an ABI GeneAmp® 9700 PCR thermocycler (ABI, CA, United States). The PCR reactions were performed as follows: initial denaturation at 95°C for 3 min, followed by 27 cycles of denaturing at 95°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 45 s, and single extension at 72°C for 10 min, and end at 4°C. The PCR mixtures contain 5 × TransStart FastPfu buffer 4 μL, 2.5 mM dNTPs 2 μL, forward primer (5 μM) 0.8 μL, reverse primer (5 μM) 0.8 μL, TransStart FastPfu DNA Polymerase 0.4 μL, template DNA 10 ng, and finally ddH2O up to 20 μL. PCR reactions were performed in triplicate. The amplicon products were extracted from 2% agarose gel and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, United States) according to manufacturer’s instructions and quantified using Quantus™ Fluorometer (Promega, United States).
Then, the amplicons were pooled in equimolar and sequenced using the Illumina MiSeq platform (Illumina, San Diego, United States) according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China).

Microbial community genomic DNA was extracted from Rumen fluid samples using the E.Z.N.A.^® soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer’s instructions. The DNA extract was checked on 1% agarose gel, and DNA concentration and purity were determined with the NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, Wilmington, USA). The hyper-variable region V3-V4 of the bacterial 16S rRNA gene was amplified with primer pairs 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) by an ABI GeneAmp^® 9700 PCR thermocycler (ABI, CA, USA). The PCR amplification of the 16S rRNA gene was performed as follows: initial denaturation at 95°C for 3 min, followed by 27 cycles of denaturing at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 45 s, and single extension at 72°C for 10 min, and end at 4°C. The PCR mixtures contain 5 × TransStart FastPfu buffer 4 μL, 2.5 mM dNTPs 2 μL, forward primer (5 μM) 0.8 μL, reverse primer (5 μM) 0.8 μL, TransStart FastPfu DNA Polymerase 0.4 μL, template DNA 10 ng, and finally ddH₂O up to 20 μL. PCR reactions were performed in triplicate. The PCR product was extracted from 2% agarose gel and purified using the AxyPrep DNA Gel Extraction kit (Axygen Biosciences, Union City, CA, USA) according to the manufacturer’s instructions and quantified using the Quantus Fluorometer™ (Promega, USA).

Microbial community genomic DNA was extracted from rumen fluid samples using the E.Z.N.A.^® soil DNA Kit (Omega Bio-Tek, Norcross, GA, U.S.) according to the manufacturer’s instructions. The DNA quality and quantity were determined by a NanoDrop 2000 UV–vis spectrophotometer (Thermo Fisher Scientific, Wilmington, USA). The V3–V4 region of the bacterial 16S rRNA gene was amplified with the primer pair 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) by an ABI GeneAmp^® 9700 PCR thermocycler (ABI, CA, USA). The PCR amplification of the 16S rRNA gene was performed as follows: initial denaturation at 95°C for 3 min, followed by 27 cycles of denaturing at 95°C for 30 s, annealing at 55°C for 30 s and extension at 72°C for 45 s, and a single extension at 72°C for 10 min, ending at 4°C. The PCR mixtures contained 4 μL of 5 × TransStart FastPfu buffer, 2 μL of 2.5 mM deoxynucleoside triphosphates, 0.8 μL of each primer (5 μM), 0.4 μL of TransStart FastPfu DNA polymerase, 10 ng of template DNA, and ddH₂O to reach a total volume of 20 μL. The PCR products were purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and quantified using a Quantus™ Fluorometer (Promega, USA). Purified amplicons were sequenced on an Illumina MiSeq PE300 platform (Illumina, San Diego, USA).