Millipore nucleofast 96 pcr kit

Total DNA extracted from root samples was subjected to molecular identification by targeting the ITS2 gene [23 (link)]. Briefly, 5 μL of each DNA sample was amplified by standard PCR, and the latter was performed using HotStar Taq polymerase according to the manufacturer's instructions (Qiagen) in a thermocycler (Applied Biosystem, Paris, France). Electrophoresis on a 1.5% agarose gel was performed to separate the SYBR safe-stained PCR products (Thermo Fisher, Bourgoin Jallieu, France), and the bands were visualized under an ultraviolet transilluminator. The PCR products were purified using the Millipore NucleoFast 96 PCR kit according to the manufacturer's recommendations (Macherey-Nagel, Düren, Germany) and then sequenced using a BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) with an ABI automatic sequencer (Applied Biosystems). The generated sequences were assembled using chromas Pro 1.7 software (Technelysium Pty Ltd., Tewantin, Australia) and compared to the NCBI database (http://blast.ncbi.nlm.nih.gov/Blastcgi) for species identification.

All colonies that remained unidentified by MALDI-TOF–MS were subjected to molecular identification by sequencing the bacterial 16S rRNA^{25 (link)} and the fungal ITS1, ITS2, β tubulin and TEF regions. The primers used in this study are reported in Table S1. DNA extraction was performed using BioRobot EZ1 (Qiagen, Les Ullis, France) using the commercial EZ1 DNA Tissue Kit according to the manufacturer’s instructions (Qiagen). PCR was performed using Hotstar Taq polymerase according to the manufacturer's instructions (Qiagen) using a thermocycler (Applied Biosystem, Paris, France). PCR products were separated by electrophoresis on a 1.5% agarose gel and stained with SYBR® safe (Thermo Fisher Scientific) before being visualized under an ultraviolet transilluminator. PCR products were then purified using a Millipore NucleoFast 96 PCR kit following the manufacturer's recommendations (Macherey–Nagel, Düren, Germany) and sequenced using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) with an automatic sequencer ABI (Applied Biosystems). Sequences were assembled using the software ChromasPro 1.7 (Technelysium Pty Ltd., Tewantin, Australia) and blasted in the NCBI databank using default settings to identify bacterial species^{26 (link)} and against the Mycobank database http://www.mycobank.org/ to identify fungal species.