Mehdi Hasan Faruq

From : Shin-ichi ITO, Shogo YANO, Shuhei TANAKA, Mitsuro KAMEYA-IWAKI The Use of Resting Spore Spheroplasts in the DNA Analysis of Plasmodiophora brassicae Japanese Journal of Phytopathology1994 Volume 60 Issue 4 Pages 491-495 https://doi.org/10.3186/jjphytopath.60.491

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"Resting spores (RSs) of P. brassicae (Hagi isolate) were extracted from homogenized root galls of Chinese cabbage (Brassica campestris Pekinensis group) cv. Kinsho No.2, washed several times with distilled water to minimize bacterial contamination, and suspended in 0.05M Tris-HCl (pH 7.6). RSs (1•~ 109) extracted were incubated in 10ml of 0.01M EDTA, 0.05M 2-mercaptoethanol, 0.05M Tris-HCl (pH 7.2) with gently shaking at 30•Ž for 30min, and then washed with 0.05M Tris-HCl (pH 7.2), spun down at 3,000•~g for 10min. The pellet was suspended and incubated in 1ml of 2% (w/v) Neutral proteinase (Nagase), 0.05M Tris-HCl (pH 7.2) at 30•Ž for 2hr with gently shaking. After washing with 0.05M Tris-HCl (pH 7.2), RSs were incubated in 1ml of 0.1% (w/v) lipase (Wako), 0.002M 2-mercaptoethanol, 0.05M Tris-HCl (pH 7.2) at 30•Ž for 2hr. RSs were then washed with MBM (0.8M MgSO4 dissolved in 0.05M sodium maleate buffer, pH 5.5), and incubated in 2ml MBM containing 4% (w/v) Novozym 234 (Novo Biolab), 0.2% (w/v) chitinase (Sigma) at 30•Ž for 24hr. Spheroplastization of RSs was examined with a Normarski differential interference contrast microscope after the exposure of RSs to low osmotic pressure. All buffer solutions used for preparing spheroplasts contained streptomycin sulfate (Meiji seika, 100ƒÊg/ml) and sefaroridine (Shionogi, 100ƒÊg/ml) to inhibit a bacterial growth. To prepare genomic DNA, the Spheroplasts (8•~108) in a 13ml tube (Assist) were spun down at 5,000•~g for 10min and incubated in 500ƒÊl of 0.5M EDTA, 1% (w/v) N-lauroyl sarcosinate (Wako), 0.4% (w/v) proteinase K (Wako) at 50•Ž for more than 5hr. To the resulting lysate 2.5ml of TE (10mM Tris-HCl, 1mM EDTA, pH 8.0) was added and the lysate was incubated at room temperature for 30min with gently shaking. Addition of 5 volume of TE to the lysates was necessary to reduce the viscosity of the lysate and essential to obtain high yield of the DNA. Potassium acetate (final concentration: 2M) was added to the lysate and the solution was kept in a ice bath for 30min. After the centrifugation (5,000•~g), 1/10 volume of 2.5M sodium acetate (pH 5.2) was added to the supernatant. DNA was precipitated by the addition of 2 volume of cold ethanol, spooled with a glass rod or spun down at 5,000•~ g, and dissolved in 500ƒÊl of TE. After the treatment of RNase (Boehringer, 40ƒÊg/ml) at 37•Ž for 2hr, the solution was subjected to the phenol extraction) and dissolved again in TE. For comparing, genomic DNA of RSs was also prepared from mechanically-disrupted RSs6). RSs were ground in liquid nitrogen with a motar and a pestle and vortexed with small glass beads in a solution containing 0.15M EDTA, 0.05M Tris-HCl (pH 8.0). Genomic DNA was purified from the solution by the same method described above. "

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