Geneamp 2700 thermal cycler

Extraction of the LMW RNA fraction containing miRNAs was obtained by a commercial column-based system (RNeasy MinElute Spin Columns, Qiagen), suitable for both cell supernatants and lysates. In our experimental conditions, 800 μL of supernatant or 700 μL of cell lysates (from 2 × 10⁶ cells) were loaded per column. Fifty nanograms of RNA were used as template for reverse transcription using the miScript II RT Kit (Qiagen) and a thermocycler system (GeneAmp 2700 Thermal Cycler, Applied Biosystems). RT-qPCR was performed using miScript SYBR Green PCR Kit (Qiagen) and Eppendorf Mastercycler ep realplex ES real-time thermocycler (Eppendorf). Normalization was obtained using the spike-in control C. elegans miR-39.

Total DNA was extracted from pure cultures of all 29 isolates by collecting 0.1 g aerial mycelia from colonies actively growing on PDA using the Power Soil DNA Isolation Kit (MO BIO Laboratories Inc., Carlsbad, CA, USA) following the manufacturer’s instructions. Polymerase Chain Reaction (PCR) was used for all isolates to amplify the internally transcribed spacer region (ITS), including the 5.8S ribosomal gene as well as part of the translation elongation factor 1 alpha gene (TEF1), using ITS1/ITS4 and TEF71F/TEF997R primers, respectively [40 ,41 (link)]. PCR reactions consisted of 13.85 µL NF H₂O, 2 µL 10× Buffer, 2 µL dNTPs (2.0 mm), 0.4 µL Blotto 10% w/v, 0.25 µL forward primer (20 µm), 0.25 µL reverse primer (20 µm), 0.25 µL DreamTaq (ThermoFisher Scientific, Burlington, ON, Canada), and 1 µL sample DNA. PCR amplifications were performed using a GeneAmp 2700 thermal cycler (Applied Biosystems, Foster City, CA, USA) under the conditions previously described [40 ,41 (link)]. All PCR products were purified using a QIAquick PCR purification Kit (QIAGEN Inc., Valencia, CA, USA), and both strands of the ITS and TEF1 were sequenced using a 8-capillary AB 3500 Genetic Analyzer Sanger Sequencer (Foster City, CA, USA) at the SuRDC.