16S sequencing of Fagus sylvatica L. deadwood
The bacterial 16S rRNA gene was amplified using barcoded 515F and 806R primers. PCR amplification was performed with 5 l of 5 Q5 Reaction Buffer (New England Biolabs), 5l of 5 Q5 High GC Enhancer (New England Biolabs) and 0.25 l Q5 High-Fidelity DNA Polymerase (New England Biolabs), 1.5 l of bovine serum albumin (10 mg/ml), 0.5 l of dNTPs Nucleotide Mix 10 mM (Bioline), 1 l of each primer, 9.75 l of H2O and 1 l of template DNA. The PCR reactions were purified with a MinElute PCR Purification Kit (Qiagen) and mixed in equimolar amounts according to concentration measured on the Qubit 2.0 Fluorometer (Thermo Fisher Scientific). Sequencing libraries were prepared using the TruSeq PCR-Free Kit (Illumina) according to manufacturers instructions and were sequenced on an Illumina MiSeq (2  250 bases).