16S rRNA gene sequencing: Crude oil contaminated soil, 6 months bioremediation treatment, replicate 1
A single-step 30 cycle PCR using HotStarTaq Plus Master Mix Kit (Qiagen, Valencia, CA) were used under the following conditions: 94 degree celsius for 3 minutes, followed by 28 cycles of 94 degree celsius for 30 seconds;  53 degree celsius for 40 seconds and 72 degree celsius for 1 minute; after which a final elongation step at 72 degree celsius for 5 minutes was performed.  Following PCR, all amplicon products (V4 regiosn) from different samples were mixed in equal concentrations and purified using Agencourt Ampure beads (Agencourt Bioscience Corporation, MA, USA).   Samples were sequenced utilizing Roche 454 FLX titanium instruments and reagents and following manufacturers guidelines