F star taq dna polymerase

Hypervariable regions (V3–V4) of the 16 S rRNA gene were amplified by PCR in a total volume of 50 μl containing 0.25 μl of BioFact F-Star Taq DNA polymerase (BioFACT™, Seoul, Republic of Korea), 20 ng of DNA template, 5 μl of 10× Taq buffer (20 mM Mg²⁺), 1 μl of 10 mM dNTP mix, and 2 μl of forward and reverse barcoded primers (10 pmol/μl). PCR was performed in a GeneAmp® PCR system 9700 (Applied Biosystems, Foster City, CA, USA) under the following thermocycling conditions: initial denaturation at 94 °C for 5 min, followed by 28 cycles of denaturation (30 s, 95 °C), annealing (30 s, 60 °C), and extension (30 s, 72 °C), a final extension step at 72 °C for 10 min, and cooling to 4 °C. PCR products were confirmed by electrophoresis in 1% agarose gels, visualized using a Gel Doc system (BioRad, Hercules, CA, USA), purified with PureLink Quick Gel Extraction and PCR Purification Combo Kit (Invitrogen, Carlsbad, CA, USA), and quantified using a Qubit 2.0 fluorometer (Invitrogen). The size of the libraries was assessed using BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA). The amplicons of participants were sequenced in an Illumina MiSeq sequencing system (Illumina, San Diego, CA, USA).

Hypervariable regions (V3-V4) of 16S ribosomal ribonucleic acid (rRNA) gene were amplified using barcoded universal primers for each sample. Polymerase chain reaction (PCR) was carried out by using BioFact F-Star taq DNA polymerase (BioFACT, Seoul, Republic of Korea). Briefly, a final volume of 50 μL of PCR reaction contained about 20 ng of DNA template, 5 μL of 10× Taq buffer (20 mM Mg²⁺), 1 μL of 10 mM dNTP mix, 2 μL of forward and reverse barcoded primers (10 pmol/μL), and 0.25 μL of DNA polymerase. PCR reactions were amplified using a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA). The PCR program was as follows: initial for 5 minutes hold at 94°C, followed by 28 cycles of denaturation (30 seconds, 95°C), annealing (30 seconds, 60°C), and extension (30 seconds, 72°C), with a final extension step (10 minutes, 72°C) followed by holding at 4°C. The PCR product was confirmed by using 1% agarose gel electrophoresis and visualized under a Gel Doc system (BioRad, Hercules, CA). The amplified products were purified with PureLink Quick Gel Extraction and PCR Purification Combo Kit (Invitrogen, Carlsbad, CA) and quantified by the Qubit 2.0 fluorometer (Invitrogen). The size of library was assessed by BioAnalyzer (Agilent Technologies, Santa Clara, CA). The amplicons were pooled and sequenced with an Illumina MiSeq sequencing system (Illumina, San Diego, CA).