Hipura fungal dna isolation kit

Fungal mycelia grown in potato dextrose broth (PDB) for more than one week were taken and fungal genomic DNA was extracted using the HiPurA fungal DNA isolation kit (Himedia) following manufacturer’s instruction. Fungal rDNA-ITS region was amplified from the extracted genomic DNA by using fungal domain specific primer ITS1F and ITS4R [18 (link)]. PCR was performed in a GeneAmp 9700 Thermal Cycler (Applied Biosystems, USA) under the following conditions, initial denaturation step at 94 °C for 5 min, followed by 30 cycles of 94°C for 1 min, 52 °C for 30 s, and 72°C for 1 min, with a final extension step of 72°C for 10 min. The PCR amplicons were excised and purified using a QIA Quick Gel Extraction Kit (Qiagen, Germany). The amplicons were sequenced in Applied Biosystems 3700 Genetic Analyser (Applied Biosystems, USA) with Big Dye Terminator ver. 3.1. The sequences were then compared with the GenBank database by the BLASTN program. Sequences obtained were then searched for similarity with other deposited sequences in GenBank. Alignments and phylogenetic analysis were performed using MEGA 4.0 software [19 ].

The fungal mycelia grown on surface of PDA were harvested, placed into 1 mL Millipore water, frozen in liquid nitrogen, and disrupted using mortar and pestle. The extraction of genomic DNA was carried out using HiPurA fungal DNA isolation kit (Himedia, Mumbai, India).

Freeze-dried fungal tissues were considered for extraction of DNA. Genomic DNA was extracted using HiPurA fungal DNA isolation kit (Himedia, Mumbai, India) and characterized by two universal primers—lTS1F (5’-TCCGTAGGTGAACCTGCGG-3’) and 1TS4R (5’-TCCTCCGCTTATTGATATGC-3’) [12 ]. Amplification was carried out in a GeneAmp 9700 thermal cycler (Applied Biosystems, Foster City, California, USA) under the following conditions: initial denaturation at 95 °C for 5 minutes; 35 cycles of denaturation at 9 °C for 1 minute, annealing at 52 °C for 30 seconds, extension at 72 °C for 1 minute; and a final extension at 72 °C for 10 minutes. The amplified Polymerase Chain Reaction products were electrophoretically analyzed on 1.5% agarose gel along with a 100 bp marker ladder. Amplified ITS ribotypes were purified using the QIAquick gel extraction kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. All the purified amplicons were sequenced (Xcelris Lab, Ahmedabad, lndia).