Barcoded Pyrosequencing of communities  in hot springs of western China with bacterial primers
PCR amplification consisted of an initial denaturation at 95ºC for 10 min, followed by denaturation at 95ºC for 45 s, annealing at 42ºC for 1 min, extension at 72ºC for 1min; 25 cycles, with the last cycle being followed by a final extension at 72ºC for 5 min. Individual reagents and their concentrations were as follows: 1× PCR buffer with 1.5 mM Mg2Cl, dNTPs (100 lM each), 0.25 lM each primer, 2.5 U of DNA polymerase (Ex-Taq) (TaKaRa, Dalian, China), and ~50 ng of total DNA. PCR products were purified using an E.Z.N.A. Gel Extraction Kit (Omega Bio-Tek Inc, USA) according to the manufacturer’s instructions.