Plant total DNA was extracted following the methods of Lin et al. (2012) (link)
via microprep buffer containing a mixture of three kinds of buffer, namely, DNA extraction buffer [0.35 M sorbitol, 1.1 M Tris and 5 mM EDTA (pH adjusted to 7.5)], nuclei lysis buffer (1.2 M Tris, 0.05 M EDTA, 2 M NaCl and 2% CTAB), and 5% sarkosyl, at a 5:5:2 volumetric ratio. The collected leaf samples (50-100 mg per sample) were ground in 750 μl of microprep buffer. The ground sample solution was incubated at 65 °C for 30-120 min. Five hundred microliters of chloroform/isoamyl (24:1) mixture was then added to the sample solution, which was then vortexed for 0.5-1 min. The homogenized samples were then centrifuged at 12000 × g (Rotor F45-24-11, Centrifuge 5415D, Eppendorf, Hamburg, Germany) for 5 min. The top layer of the centrifuged suspension was pipetted (500 μl) into a new 1.5 ml Eppendorf tube. Afterward, 500 μl of isopropanol was added to the tube, after which the contents were mixed gently. The samples were pelleted by centrifugation at 12,000 × g (Rotor F45-24-11, Centrifuge 5415D, Eppendorf) for 5 min. The DNA pellet was subsequently washed with 70% ethanol and then dried in an oven for 1-2 min to remove ethanol. The DNA pellet was subsequently resuspended in 50 μl of sterilized distilled water and kept at -20 °C for further analysis.
via microprep buffer containing a mixture of three kinds of buffer, namely, DNA extraction buffer [0.35 M sorbitol, 1.1 M Tris and 5 mM EDTA (pH adjusted to 7.5)], nuclei lysis buffer (1.2 M Tris, 0.05 M EDTA, 2 M NaCl and 2% CTAB), and 5% sarkosyl, at a 5:5:2 volumetric ratio. The collected leaf samples (50-100 mg per sample) were ground in 750 μl of microprep buffer. The ground sample solution was incubated at 65 °C for 30-120 min. Five hundred microliters of chloroform/isoamyl (24:1) mixture was then added to the sample solution, which was then vortexed for 0.5-1 min. The homogenized samples were then centrifuged at 12000 × g (Rotor F45-24-11, Centrifuge 5415D, Eppendorf, Hamburg, Germany) for 5 min. The top layer of the centrifuged suspension was pipetted (500 μl) into a new 1.5 ml Eppendorf tube. Afterward, 500 μl of isopropanol was added to the tube, after which the contents were mixed gently. The samples were pelleted by centrifugation at 12,000 × g (Rotor F45-24-11, Centrifuge 5415D, Eppendorf) for 5 min. The DNA pellet was subsequently washed with 70% ethanol and then dried in an oven for 1-2 min to remove ethanol. The DNA pellet was subsequently resuspended in 50 μl of sterilized distilled water and kept at -20 °C for further analysis.