Nanodrop nd 1000 device

The hypervariable V3-V5 region of the 16S rRNA gene was amplified using the primer set 341F (5′ CCTACGGGAGGCAGCAG 3′) and 907R (5′ CCGTCAATTCCTTTRAGTTT 3′). A unique 8-nucleotide barcode was added to both primers for multiplexed pyrosequencing using barcrawl (Pourmand et al., 2002 (link); Frank, 2009 (link)). Each 20-μL PCR reaction consisted of 4 μL of 5 × Phusion HF Buffer (M0530S New England BioLabs Inc.), 1.6 μL of dNTPs (2.5 μM each), 1 μL of each primer (10 μM), 0.6 μL of DMSO, 10 ng of template DNA, 0.2 μL of Phusion^® High-Fidelity DNA Polymerase (0.4 units) and 10.6 μL of pure water. PCR was performed with a thermal cycler (Bio-Rad, USA) using the following program: an initial denaturation at 98°C for 1 min; 25 cycles at 98°C for 10 s, 60°C for 30 s and 72°C for 20 s; and a final extension at 72°C for 5 min. The PCR products were detected by agarose gel electrophoresis, and the purified concentrations were measured using a NanoDrop ND-1000 device (Thermo Fisher, USA). Equal amounts of the barcoded amplicons were mixed and pyrosequenced using a ROCHE 454 FLX Titanium platform.

DNA manipulations were performed according to standard protocols (Sambrook et al., 1989 ). Genomic DNA from yeast cells was isolated according to Hoffman and Winston (1987) (link), while plasmid DNA from E. coli was isolated using the GenElute^TM Plasmid Miniprep Kit (Sigma). DNA quantity and quality were evaluated electrophoretically and spectrophotometrically using a NanoDrop ND-1000 device (Thermo Scientific, Waltham, MA, United States). Zymoclean^TM Gel DNA Recovery and DNA Clean & Concentrator^TM-5 Kits (Zymo Research, Orange, CA, United States) were used for the isolation of DNA fragments from agarose gels and for PCR amplicons purification, respectively. Long PCR amplifications were carried out with rTAQ DNA polymerase (Takara Bio, Shiga, Japan) according to manufacturer’s instructions. For colony PCR 1 μl of DNA extracted with lithium acetate-SDS method (Lõoke et al., 2011 (link)) was amplified with DreamTaq polymerase (Thermo Scientific, Waltham, MA, United States) according to the manufacturer’s instructions in 20 μl reaction volume. All PCR amplifications were carried out in a T100 Thermal cycler (Bio-Rad, Hercules, CA, United States). All primers used in this study are listed in Supplementary Table S1.