Isoplant 2 dna extraction kit

Two strains of C. auris, JCM 15448 and TWCC 58191, were previously isolated in Japanese hospitals [18 (link),19 (link)]. Ascomycetous yeast strains, isolated from otorrhea of patients with chronic otitis media in Japan from 1997 to 2017 (Table 1), were cultured using Sabouraud dextrose agar (Becton Dickinson, Baltimore, MD, USA) for 2 days at 35°C. For identification of these clinical isolates, DNA extraction was performed using Isoplant II DNA extraction kit (Nippon Gene inc., Tokyo, Japan), direct-sequencing of the 26S D1/D2 domain and ITS region, and a BLASTN homology search against known sequences.

Polymerase chain reaction-denatured gradient gel electrophoresis (PCR-DGGE) was performed to observe the culture-based bacterial communities on the leaves of the two Sphagnum mosses. First, genomic DNA was extracted from the medium after the N₂O assay using an Isoplant II DNA Extraction kit (Nippon Gene, Toyama, Japan). The PCR steps and conditions were as follows: PCR denaturation for 5 min at 95°C, and 30 cycles of amplification (15 s at 95°C, 30 s at 55°C, 30 s at 72°C), and 10 min elongation at 72°C. Then PCR products for DGGE were obtained by using the common 16S rRNA primers GC-341F (CGC CCG CCG CGC CCC GCG GGG GTC CCG CCG CCC CCG CCC GCC T AC GGG AGG CAG CAG) and 907R (CCG TCA ATT CCT TTR AGT TT) (Ferris et al., 1996 (link)) and run on a 30–70% denatured gradient gel (6% w/v). The sequences of DGGE-cutting bands were obtained using an ABI prism^TM 310 Genetic Analyzer and retained in the NCBI (BioProject No. PRJNA681491).