CTAB DNA extraction

The fresh root tissue was weighed and then immediately flash frozen in liquid nitrogen and ground to fine powder using a mortar and pestle. A 2X CTAB solution [2% (w/v) hexadecyltrimethylammonium bromide (CTAB), 100 mM Tris (pH 8.0), 20 mM EDTA (pH 8.0), and 1.4 M NaCl was autoclaved sterile, to which 2% (w/v) of polyvinylpyrrlidone (M_w 40,000) and 1% 2-mercaptoethanol are added] was preheated to 65°C and applied to the root powder at a volume of 5:1 (i.e. 5 mL 2X CTAB per 1 g of fresh root weight) and vortexed for 1 minute. The tube was incubated in a 65°C water bath for 30 minutes. After incubation, the tube was vortexed again for 10 seconds to homogenize the solution, and then a 1 mL aliquot was transferred to a 2 mL tube. One volume of chloroform:isoamyl alcohol (24:1) was added to the supernatant and vigorously shaken to form an emulsion, followed by centrifugation at 14,000 rpm for 2 minutes. This step was repeated once. The supernatant was transferred to a 1.5 mL microfuge tube and 0.7 volumes of isopropanol were added and mixed by inversion to precipitate the DNA. The tube was centrifuged at 14,000 rpm for 5 minutes. The DNA pellet was washed once with 70% ethanol and once with 100% ethanol, being centrifuged at 14,000 rpm for 2 minutes each time. The ethanol was decanted, and the pellet allowed to dry for 10 minutes on the bench top. The pellet was resuspended in 50 μL of 1X TE buffer pH 8.0.