Taq 2x master mix red

The mouse tail was digested in NTES buffer [100 mM NaCl, 50 mM Tris (pH 8.0), 50 mM EDTA, 1% SDS] with 200 µg/mL Proteinase K (1.24568.0500, Millipore) and then incubated at 55 °C overnight. Genomic DNA was extracted by phenol/chloroform and precipitated by 100% ethanol. The DNA pellet was resuspended in deionized water to a final concentration of 50–100 ng/µL for further genotyping. Genotyping was performed using Taq 2x Master Mix RED, 1.5 mM MgCl₂ (5200300-1250, Ampliqon) or 2X RBC SensiZyme™ HotStart Taq Premix (RT008, RBCBioscience), and then, the PCR products were electrophoresed on 2 or 0.8% (w/v) agarose gels with Nancy-520 (01494, Sigma) and observed under ultraviolet light.

Soil DNA was isolated with the PowerSoil® DNA Isolation Kit (MO BIO Laboratories, Inc., USA). A 789F primer (5′-TAG ATA CCC SSG TAG TCC - 3′) and 1053R nucleotide reverse primer (5′- CTG ACG RCR GCC ATG C-3′) were used to amplify the target V4-V6 region of 16S-rRNA. The primer pair was chosen because of its wide coverage of bacterial lineages [66 (link), 67 (link)]. For each sample, PCR was performed in eight to 12 tubes. The reaction volume was 50 μL. Each reaction volume consisted of 1 × Taq 2x Master Mix Red (Ampliqon, Denmark), 0.1 μM of each primer, and 10 ng template DNA. PCR conditions were 95 °C for 5 min; 27 cycles of 95 °C for 30 s, 50 °C for 30 s,72 °C for 50 s, and a final extension at 72 °C for 10 min. PCR products were visualized using 1.2% agarose gel electrophoresis and purified using a Gel/PCR DNA extraction Kit (Geneaid, Taiwan). The purified PCR products were precipitated and concentrated with isopropanol. The DNA concentration was quantified using a Qubit® 2.0 Fluorometer (Invitrogen, USA).