Metagenomic Analysis of Cactus-Olive Interface
After harvest and grinding, three fresh samples from each block of five pots were weighted and stored in 2mL eppendorf tubes at -80C. Total genomic DNA was extracted from 0.5g of powder soil and root samples using DNeasy PowerSoil Kit (QIAGEN, Hamburg, Germany) employing the manufacturers protocol. The purity and concentration of DNA was determined in 1% agarose gel and Nanodrop (Denovix, USA). The hyper variable regions V3-V4 of 16S rRNA gene were amplified with primer pairs 341F (5-CCTACGGGNGGCWGCAG-3) and 806rB (5-GGACTACNVGGGTWTCTAAT-3). The PCR was performed in triplicate in 25L mixture containg 12.5L of buffer mix, 0.5 L of each primer 0.5L of enzyme mix, 10ng of DNA template and 1L of nuclease free water. PCR was carried out following: initial denaturation at 95C for 15 min ; 20 cycles of 98C for 30s, 55C for 30 s and 72 C for 5min. PCR products were purified with AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, CA, USA) and pooled in equimolar ratios. Library preparation and Illumina MiSeq sequencing were performed at GenXPro (Frankfurt am Main, Germany)