Nanodrop 2000

A. tubingensis G131 was isolated from a French Mediterranean vineyard, classified into Aspergillus section Nigri (morphological characterization) and described as non-ochratoxigenic (biochemical characterization) [25 (link)]. The strain was cultured 3 days at 28 °C under shaking condition 120 rpm on PDB medium. The fungal mycelial mat was harvested and ground into a fine powder with liquid nitrogen and conserved at − 80 °C until used. Genomic DNA was extracted from this powder with a protocol adapted from [53 (link)]. 150 mg of mycelial powder was transferred to a pre-cooled Eppendorf tube with 700 μl of CTAB solution (1% CTAB; 100 mM NaCl; 100 mM EDTA pH 8; 20 mM Tris-Cl pH 8), homogenized by vortex and incubated 1 h on ice. Genomic DNA was then extracted using two successive Phenol/Chloroform/Isoamyl Alcohol (Sigma) washes, precipitated with anhydrous ethanol and suspended in nuclease free water. Genomic DNA concentration and quality were estimated using Nanodrop 2000 (Eppendorf).

RNA was extracted from allantoic fluid infected with the wild type NDV strain IBS025/13 using Trizol LS^® reagent (Invitrogen, Carlsbad, USA) following the instructions of the manufacturer. To synthesize cDNA, 0.7–1µg of the extracted RNA was reverse transcribed using a Sensifast^® cDNA synthesis kit (Bioline, UK) according to the recommended protocol provided in the kit. The complete open reading frames of the viral NP, P, and L genes were amplified from the cDNA template and then subcloned into pCIneo to generate helper plasmids (pCIneo-NP, pCIneo-P and pCIneo-L). All cloning experiments were verified using colony PCR, restriction endonuclease digestion, and DNA sequencing. Purity and concentrations of each recombinant plasmid construct were determined using Nanodrop 2000 (Eppendorf, Germany).