HomeInternational Rice Research Notesvol. 22 no. 2 (1997)

A Rapid Method of Isolation of Genomic DNA from Filamentous Fungi

P Kachroo | S A. Leong | B B. Chattoo

Discipline: Agriculture

 

Abstract:

This method uses fungus cultured on potato dextrose agar at 28 ºC. When mat growth was visible on at least half of the plate (2-3 d), a 0.5 × 0.5 cm mycelial piece was removed with a surgical blade and placed in a 1.5-mL microfuge tube. The block was suspended in 600 μL of buffer (Tris 100 mM, pH 8.0, EDTA 150 mM, pH 8.0; NaCl 100 mM; SDS 2%; phenol 50%), and 200-300 mg of glass beads added, the contents vortexed for 3 min, and centrifuged  at 13 K for 10 min. The supernatant was treated with RNase A (10-15 μg) for 15 min at 37 ºC, extracted with phenol: chloroform: isoamyl alcohol (25:24:1), and the DNA precipitated with 2 volumes of absolute alcohol at room temperature. The DNA was washed with 70% alcohol and suspended in 30 μL of TE (Tris 10 mM; pH 8.0; EDTA 1 mM, pH 8.0). Genomic DNA appeared as a single, high-molecular-weight band when analyzed by a 0.8% agarose gel run in 0.5 × TBE.