Anthurium Leaves replicate 3
Liquid nitrogen was applied to 2 g disinfected plant tissue and ground with a sterile mortar. The ground tissues were added to 15 mL propylene tubes and 2 mL Tris-HCl 10 mM pH 8 plus 160 uL lysozyme 10 mg mL-1. The mixture was kept at 37º for 1 h. The cells were lysed with 2 mL sodium dodecyl sulfate (SDS) 10% (w/v). Sterile glass beads were added and mixed on a Vortex for 10 min. Proteins were eliminated and DNA precipitated as described by Navarro Noya et al. (28). Substrate (1.5 g) and rhizosphere DNA (1.5 g soil adhered to the roots) were extracted with three different methods (0.5 g was used per technique) as described before (32).   Primers 8-F and 556-R were used to amplify the variable regions V1-V3 of the 16S rRNA bacterial genes. The PCR mixture and the thermal cycling were done as described by Navarro-Noya et al. (28). All samples were amplified in triplicate, pooled in equal concentrations, and purified using the DNA clean and concentrator purification kit as recommended by the manufacturer (Zymo Research, Irvine, CA, USA). Quantification of the PCR products was done using PicoGreen dsDNA assay (Invitrogen, Carlsbad, USA) and a NanoDropTM 3300 fluoroespectrometer (Thermo Scientific NanoDrop). Amplicon-libraries were sequenced with a Roche GS-FLX Titanium 454 pyrosequencer (Roche, Mannheim, Germany) by Macrogen Inc. (DNA Sequencing Service, Seoul, Korea).