Picogreen fluorescent dye

Fresh needles were collected from 10 Norway spruce (Picea abies [L.] Karst.) grafted trees sampled in a 27-year old breeding orchard located north of Quebec City (Natural Resources Canada). All trees originated from central Europe, six of them being representative of distinct natural populations from Poland (3), Belorussia (1), and Latvia (2), and the four remaining ones being of unknown location. No permit was required to collect tissue in any location sampled in this study. DNA was isolated from needles using the Qiagen DNeasy Plant Mini Kit (Mississauga, ON, Canada) and quantified using the PicoGreen fluorescent dye (Invitrogen). Afterward, DNA samples were assembled in two pools of five individuals with equimolar concentrations [84 (link)]. In order to generate a reference sequence assembly with minimum genetic polymorphism, DNA was also extracted from a haploid megagametophyte, followed by whole-genome amplification using the WGA2 kit (Sigma-Aldrich, Oakville, ON, Canada).

HAdV-C5 vector alone (6 x 10⁹ pp), IVIg (10 μg) or IC-HAdV were exposed to pH 5 (sodium acetate), pH 6 (MES), pH 7 (HEPES) or pH 9 (carbonate buffer) buffers (15 μl final volume) and capsid disassembly was assessed by DNA detection using Picogreen fluorescent dye (Invitrogen). The ABI prism 7900HT PCR machine (Applied Biosystems) was programmed to measure fluorescence (λex = 488 nm and λem = 520 nm) every 2.5°C from 30 to 70°C.

Genomic DNA was extracted from replicate samples or pooled replicates (Table S1, sampling replicates and sequencing replicates) using a DNeasy PowerFood microbial kit (Qiagen, Hilden, Germany). Extracted DNA samples were amplified and sequenced at the University of Connecticut Microbial Analysis, Resources, and Services Center using the standard protocol. DNA was quantified using Pico-Green fluorescent dye (Invitrogen, Waltham, MA, USA) and was used as the template to amplify the bacterial 16S rRNA V4 region (515F and 806R) and fungal internal transcribed spacer regions (ITS3 and ITS4). Primers with Illumina adapters and dual barcodes were used for amplification. PCR conditions for bacteria consisted of 94°C for 2 min, 30 cycles of 30 s at 94°C, 30 s at 53°C, and 60 s at 72°C, and a final extension at 72°C for 5 min. PCR conditions for fungi consisted of 95°C for 2 min, 5 cycles of 30 s at 95°C, 60 s at 48°C, and 60 s at 72°C, 25 cycles of 30 s at 95°C, 60 s at 55°C, and 60 s at 72°C, and a final extension at 72°C for 5 min. PCR products were cleaned using Mag-Bind RxnPure plus (Omega Bio-tek, Norcross, GA, USA) and sequenced on Illumina MiSeq using the 2 × 250-bp kit (Illumina, Inc., San Diego, CA, USA). Negative extraction controls were also sequenced to test for contamination.

Calcium contents of cell were quantified by colorimetric endpoint assay based on the complexation of Ca²⁺ with Arsenazo III molecule at 1:2 ratio to a blue-purple product. Aliquots of 100 μl of Arsenazo III solution (Pointe Scientific, Canton, MI) were added to the wells and incubated for 10 min at room temperature. After removal of the solution, aliquots of 100 μl of 0.6 M HCl were added to the wells for 2 h at room temperature. The dissolved mineral in the HCl solution was quantified spectrophotometrically at 650 nm. A standard dilution series of calcium standard (Merck, Billerica, MA) ranging from 0 to 160 μg/ml was prepared and quantified spectrophotometrically at 650 nm. The amount of DNA in the wells were determined with Picogreen fluorescent dye (Molecular Probes, Eugene, OR) according to the manufacturer’s instructions. Calcium content was expressed as micrograms of Ca²⁺/μg DNA.

Fecal genomic DNA extraction, amplification of the V3 region, and pyrosequencing of PCR amplicons were performed as described previously32 (link). Genomic DNA was extracted from fecal samples collected from three groups of mice at age 4, 12 and 24 weeks using bead beating and an InviMag^® DNA kit (Invitek, Berlin, Germany). The V3 region of the 16S rRNA gene was amplified from each sample with the extracted fecal DNA as the template in PCR with 20 cycles. The bacterial universal primer pair for the V3 region consisted of the forward primer 5′-NNNNNNNNCCTACGGGAGGCAGCAG-3′ and the reverse primer 5′-NNNNNNNNATTACCGCGGCTGCT-3′. NNNNNNNN was the sample-unique DNA bar code of eight nucleotide sequences. The PCR amplicons from each sample were quantified with PicoGreen fluorescent dye (Thermo Fisher Scientific, Sunnyvale, USA) by using SpectraMax M5 microplate reader (Molecular Devices, San Francisco, USA), and mixed in equal ratios. The 454 sequencing adapters were ligated to the mixed PCR amplicons by blunt-end ligation. Pyrosequencing was performed on a Genome Sequencer FLX platform (Roche Diagnostics, Mannheim, Germany).