title: Figure 4 antibiotic treated
design: The PCR primer sequences, the conditions for amplification of the V3   region of the 16S ribosomal RNA gene, and multiplexed DNA sequencing   strategy were as described (Bartram et al., 2011) unless otherwise   indicated.  High-pressure liquid chromatography purified PCR primers   for the V3 region were obtained from Integrated DNA Technologies   (Coralville, IA, USA).  Bacterial DNA was amplified using an AccuPrime   PCR Kit (Invitrogen Life Technologies, Grand Island, NY, USA) on a   Techne Genius Model FGEN02TP Thermal Cycler using the following   conditions: 95°C for 6 minutes denature; 95°C for 2 minutes, 50°C for 2   minutes, 72°C for 2 minutes 30 cycles; 72°C for 4 minutes extend. Each   reaction contained 1 U Taq polymerase, 5 µl 10x buffer 1(600 mM Tris-  SO 4  (pH 8.9), 180 mM (NH 4 )2SO 4 , 20 mM MgSO 4 , 2 mM dGTP, 2 mM   dATP, 2 mM dTTP, 2 mM dCTP, thermostable AccuPrimeTM protein, 10%   glycerol), 20 µM forward primer, 20 µM reverse primer, and up to 60 ng   DNA in a total volume of 50 µL. PCR reactions were performed in   triplicate and reaction products were pooled prior to purification.