A plant with novel insertion of ONSEN
Plant leaves weighing approximately 200 mg were homogenized in liquid nitrogen using a mortar and pestle and then divided into equal portions for total DNA and RNA isolation. Total DNA was isolated using a modified CTAB protocol [76]. Briefly, 0.5 mL of CTAB1 buffer, preheated to 75 C and containing 6% -mercaptoethanol and 0.5% polyvinylpyrrolidone, was added to the frozen powder. The lysate was transferred to a 1.5 mL tube and incubated at 75 C for 1 h. After cooling, an equal volume of chloroform was added, and the mixture was centrifuged. The upper aqueous phase was transferred to a new 1.5 mL tube containing two volumes of CTAB2 buffer. The resulting pellet was resuspended in 0.2 mL of 1 M NaCl, and DNA was precipitated with an equal volume of isopropanol, followed by centrifugation. The pellet was washed with 70% ethanol and resuspended in nuclease-free water. RNase treatment was performed, followed by iso-propanol reprecipitation and ethanol washing. The final DNA pellet was resuspended in nuclease-free water for downstream analyses.