A 5 cm segment of B. distachyon leaf was ground up into a powder by a Verder ball grinder after being frozen in liquid nitrogen. 600 µL of DNA extraction buffer (100 mM sodium chloride, 50 mM Tris, 25 mM ethylene diamine tetra-acetic acid (EDTA), 1% sodium dodecyl sulfate (SDS), 10 mM beta-mercaptoethanol) was then added and mixed to break up the cells and prevent DNA degradation. The tube was incubated in a 65°C hot block for 10 minutes then iced. Next, 250 µL of 5M potassium acetate was added and mixed, and the tube was incubated on ice for 20 minutes for the carbohydrate and proteins to precipitate. The tube was centrifuged at maximum speed for 10 minutes, and the supernatant containing the DNA was transferred into a new tube containing 600 µL of 100% isopropanol. This solution was mixed, and the DNA was allowed to precipitate before centrifuging the tube at maximum speed for 15 minutes. The supernatant was removed, and the pellet containing the extracted DNA was rinsed with 300 µL of 70% ethanol and centrifuged for another 10 minutes. The liquid was pipetted out, and the remaining ethanol was allowed to dry completely. Lastly, the tube had 50 µL of T10E1 (10 mM Tris, 1 mM EDTA) added to it, which preserves the DNA, and was incubated at 65°C for 10 minutes. The pellet was mixed into the solution by pipetting up and down.
Recent comments