Gs flx titanium lv empcr kit

Fragments of bacterial and archaeal 16S rRNA genes were amplified using the primers B27f/B533r (B27f: 5′-AGAGTTTGATCMT GGCTCAG-3′, B533r: 5′-TTACCGCGGCTGCTGGCAC-3′) and A344f/A915r (A344F: 5′-ACGGGGYGCAGCAGGCGCGA-3′, A915R: 5′-GTGCTCCCCCGCCAATTCCT-3′), respectively, with Roche 454 sequencing adapters (Table S1) (44 ). The first PCR reaction (20 μL) contained 2.5 U of Ex Taq DNA polymerase (Takara, Otsu, Japan), 2 μL of 10×buffer, 1.6 μL of dNTPs, 0.4 μL of each primer, 1 μL of bovine serum albumin (BSA), 1 μL of template DNA, and 13.1 μL of water. The PCR conditions used for bacterial amplification were as follows: 5 min at 95°C; 26 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and primer extension at 72°C for 2 min and a final extension at 72°C for 10 min. PCR conditions for archaea were as follows: 94°C for 4 min, 25 cycles of 94°C for 1 min, 52°C for 1 min, and 72°C for 2 min, and a final extension at 72°C for 7 min. Purified PCR products were quantified by fluorimetry with a Qubit^® dsDNA HS Assay kit (Invitrogen, Waltham, MA, USA) and were then pooled and sequenced using a GS FLX Titanium LV emPCR Kit on a GS FLX+ Instrument (Roche, Indianapolis, IN, USA) according to the manufacturer’s instructions.