Pf mag bind stool dna kit

Total microbial genomic DNA was extracted from stool and tissue samples using the PF Mag-Bind Stool DNA Kit (Omega Bio-tek, Georgia, U.S.) according to manufacturer’s instructions. The quality and concentration of DNA were determined by 1.0% agarose gel electrophoresis and a NanoDrop® ND-2000 spectrophotometer (Thermo Scientific Inc., USA) and kept at -80 °C prior to further use. The hypervariable region V3-V4 of the bacterial 16 S rRNA gene were amplified with primer pairs 338 F (5’-ACTCCTACGGGAGGCAGCAG-3’) and 806R(5’-GGACTACHVGGGTWTCTAAT-3’) by an ABI GeneAmp® 9700 PCR thermocycler (ABI, CA, USA). The PCR reaction mixture including 4µL 5×Fast Pfu buffer, 2µL 2.5 mM dNTPs, 0.8µL each primer (5µM), 0.4µL Fast Pfu polymerase, 10 ng of template DNA, and ddH2O to a final volume of 20 µL. PCR amplification cycling conditions were 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. All samples were amplified in triplicate. The PCR product was extracted from 2% agarose gel and purified. Then quantified using Quantus™ Fluorometer (Promega, USA).