3500 automated sequencer

DNA from the grown colonies was isolated using commercially available PROBA-GS kit (DNA Technology, Russia). Polymerase chain reaction was performed with an Eppendorf MasterCycler Personal cycler. Each PCR reaction mixture contained 2.5 µl of 10× reaction buffer, 1 µl of 10 mM dNTPs, 1 µl of 10 µM forward primer, 1 µl of 10 µM reverse primer, 3 µl of 25 mM Mg²⁺, 1 µg of template DNA, 2.5 units of thermostable Taq DNA polymerase (Evrogen, Russia), and deionized water (up to 25 μl). PCR regime included initial denaturation at 94 °C for 5 min; 35 cycles of denaturation at 94 °C for 30 s, annealing at 54 °C for 30 s, elongation at 72 °C for 45 s; final elongation at 72 °C for 10 min. Fungal-specific primers for molecular identification were: direct ITS1 primer—5′-TCCGTAGGTGAACCTGCGG; reverse ITS4 primer—5′-TCCTCCGCTTATTGATATGC (White et al. 1990 ).
PCR products were stained with ethidium bromide and visualized at 312 nm with a TCO-20LM transilluminator after electrophoresis in 2% agarose gel.
RCR products were purified from the gel with a Cleanup Standard kit (Evrogen, Russia) and sequenced with an Applied Biosystems 3500 automated sequencer using a BigDye Terminator v3.1 Cycle Sequencing Kit and ITS1/ITS4 primers.

DNA was extracted from fin tissues using the Promega Wizard DNA extraction kit. Samples were screened at 17 microsatellite loci (Supporting Information Table 1). PCR was performed in a volume of 10 μl PCR solution consisting of 1 μl DNA (~ 5 ng), 5 μl Mastermix and 4 μl primer mix (10 mM). PCR amplifications were conducted on BioRad MyCycler thermal cycler, with conditions consisting of one denaturation step of 15 min at 95°C, followed by 35 cycles of 30 s at 94°C, 90 s at 57°C and 1 min at 72°C, followed by a final extension step of 30 min at 60°C. PCR products were sized on an ABI 3500 automated sequencer against a LIZ 500 size standard using GeneMapper 4.1 (Applied Biosystems).