Preparation of Megabase-size DNA from Plants
Procedure
1) Grind 30-50 g of the fresh or frozen tissue into powder in liquid nitrogen with a mortar and pestle (do not over grind) and immediately transfer into an ice cold 500 ml beaker containing 200 ml ice-cold wash buffer (HB) plus 0.15% beta-mercaptoethanol (add before use) and 0.5% Triton X-l00 (20% stock in HB).
2) Repeat step 1, 4-6 times in order to obtain enough nuclei to embed.
3) Swirl each mixture from step 1 with a magnetic stir bar for 20 minutes on ice.
4) Filter the mixture from step 3 into an ice-cold 250 ml centrifuge bottle through two layers of cheesecloth and one layer of miracloth (Calbiochem order # 475855, USA) placed in a single large, wide mouth funnel. To obtain more nuclei, remove the cheesecloth and squeeze the remainder of the homogenate into the centrifuge bottle.
5) Pellet the filtered homogenate by centrifugation in a fixed-angle rotor at 1,800 g at 4 degrees C for 20 minutes (about 3,500 rpm in GSA Rotor)
6) Discard the supernatant fluid and gently resuspend the pellet in 1-5 ml of ice cold wash buffer with assistance of a small paint brush. After resuspension adjust the volume to 30 ml with wash buffer and transfer to a 50 ml round bottom oakridge tube.
7) Pellet the nuclei by centrifugation at 1,800 g, 4 degrees C for 15 minutes in a swinging bucket centrifuge (about 3,000 rpm in HS-4 rotor Sorvall).
8) Wash the nuclei pellet 2 additional times by resuspension in wash buffer followed by centrifugation at 1,800 g, 4 degrees C for 15 minutes. During the last washing step the resuspended nuclei are filtered through one layer of miracloth by gravity into new oakridge tubes.
9) After the final wash, resuspend the pelleted nuclei in a
small volume of HB (2 to 3 ml). Count the nuclei with a hemacytometer
under a phase contrast microscope and adjust to approximately
4 x 10^7 nuclei/ml with addition of HB (for the plants with haploid
genomic sizes of 500-1,000 Mb).
Notes
In practice, the optimum number of nuclei depends on the genomic
size of the plant and for some plants species it can be hard to
count the nuclei. When preparing nuclei for the first time we
optimize the nuclei concentration empirically. We suggest preparing
the nuclei as described above and in the final step embed 4 different
concentrations of nuclei (e.g., 1 ml concentrated nuclei + 3 ml
of HB, 2 ml concentrated nuclei + 2 ml of HB, and 4 ml concentrated
nuclei + 0 ml of HB).
Solutions