Illustra exoprostar kit

From all yeast isolates the genomic DNA was extracted with the Wizard Genomic DNA Purification Kit (Promega), quantified and frozen at −20 °C until later use. Yeast isolates were identified by PCR analysis of the internal transcribed spacer region (ITS), which comprise the 5.8S rRNA and two flanking regions (ITS1 and ITS2), with the oligonucleotides ITS1 and ITS4 [37 (link)]. PCR products were analyzed by electrophoresis on 1.5% agarose gel. Results obtained with the ITS-PCR analysis were confirmed by Sanger-sequencing of the ITS region. For this, the ITS-PCR product was first purified with an Illustra ExoProStar kit (GE Healthcare Life Sciences, Pittsburgh, EUA), and sequencing reactions were prepared with the BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, Califórnia, EUA). Sequencing reactions were purified with BigDye XTerminator (Applied Biosystems, Foster City, Califórnia, EUA) and analyzed in the 3500 Genetic Analyzer (Applied Biosystems, Foster City, Califórnia, EUA). Sequence reactions were analyzed with BioEdit software and with the Basic Local Alignment Search Tool (BLASTn-NIH). From a total of 568 isolates, 500 were identified as non-Saccharomyces species.

The yeast isolates were submitted to ITS-PCR as described by Kurtzman et al. [16 (link)]. These reactions were carried out in 0.2-mL microtubes containing a mixture of 6.7 µL Milli-Q water, 10 µM forward primer and 10 µM reverse primer, 12.5 µL GoTaq^®Green Master Mix, and 3 µL DNA. The efficiency of the amplifications was monitored by electrophoresis (60 min, 80 V) on 1.5 % agarose gel prepared in 1× TBE buffer and stained with SYBR^® Safe. Strains previously identified at the Laboratory of Fungal Biology, Department of Microbiology and Immunology, IBB, were used as controls. The amplified fragments were purified using the Illustra™ ExoProStar™ Kit (GE Healthcare Life Sciences).

We purified amplified DNA by using an Illustra ExoProStar Kit (GE Healthcare Life Sciences, https://www.gelifesciences.com). We sequenced double-stranded DNA directly by using the Sanger chain-termination method and the BigDye Terminator v3.1 Cycle Sequencing Kit Protocol and the ABI PRISM 3700 DNA Analyzer (Applied Biosystems, https://www.thermofisher.com). We used sequencing primers CrCon2+ and CrCon2–, the same used in the nested PCR (5 (link)). We assembled consensus sequences of each segment and analyzed them by using the SeqMan Program in the Lasergene Package (https://www.dnastar.com).