nuclear DNA from tomato. This protocol includes the use of reagents which inhibit oxidation of polyphenols
and/or absorb polyphenolic compounds, as well as the nuclear-stabilizing agent 2-methyl-2,4-pentanediol (see
Watson and Thompson 1986 for a review).
Because seedlings possess higher concentrations of nuclei per gram of tissue than do leaves or other
adult tissues, we were interested in developing a DNA isolation technique that would work with tomato seedlings.
To avoid the phenolic compounds associated with seed coats still attached to seedlings, we developed a simple
technique for growing large batches of seedlings hydroponically and quickly removing the seed coats during
harvest. Briefly, styrofoam pellets were arranged in 5 rows on the bottom of a plastic greenhouse tray (53.5 x 27 x
6 cm). A piece of fiberglass window screen was cut to fit in the tray and placed on top of the styrofoam pellets. A
second plastic tray with a 1 inch diameter hole cut in the bottom was then placed in the first tray to hold the pellets
and window screen in place. Water was poured through the hole in the top tray. Once the bottom tray had become
2/3 full, the top tray was removed, and tomato seeds were placed on top of the floating window screen at a density
of 1-3 seeds/cm2. Trays containing seeds were stored at 20°C for 2 weeks (until seedlings were approximately 20
cm in length). Each screen was lifted from the water surface and turned upside down so that roots extended
upward. Seedlings were pulled gently by their roots through the window screen leaving the seed coats behind.
Decoated seedlings were immediately submerged in ice water. In a given DNA isolation, 200-500 g of seedlings
from 10-20 trays were harvested.
When leaves were used for DNA isolation, young leaves were gently picked from plants with forceps. After
removing a leaf, it was immediately submerged in ice water. Approximately 200-500 g of leaves were collected for
one isolation.
To isolate tomato nuclei, seedlings/leaves were removed from ice water, placed in 1000 ml of ice cold
ethyl ether for three minutes. Ether removes waxes and makes cells more friable. The seedlings/leaves were
removed from the ether, washed 4 times in 4°C TE buffer (10 mM Tris, 1 mM EDTA, pH 7.0), and placed in 3000
ml of ice cold extraction medium (1.0 M 2-methyl-2,4-pentanediol, 10 mM PIPES-KOH, 10 mM MgCl2, 2%
polyvinylpyrrolidone, 10 mM sodium metabisulfte, 5 mM 2-mercaptoethanol, 0.5% sodium diethyldithiocarbamate,
pH 6.0). The seedlings/leaves were then homogenized in a Waring commercial blender (high speed for 30
seconds), squeezed through muslin, and filtered again through 20 µm nylon mesh. To lyse mitochondria and
plastids, Triton X-100 was added to the filtrate to a final concentration of 0.5% (Watson and Thompson 1986). The
mixture was placed into approximately twelve 250 ml centrifuge bottles and spun at 1200 x g for 20 min at 4°C.
Supernatants were decanted, and each pellet was resuspended in 0.5 ml MPD buffer (0.5 M 2-methyl-
2,4pentanediol, 10 mM PI PES-KOH, 10 mM MgCl2, 0.5% Triton X-100, 10 mM sodium metabisulfite, 5 mM 2-
mercaptoethanol, pH 7.0). The nuclear suspension was halved, and each half (about 5 ml each) was layered onto
one of two 15 ml beds of 50% Percoll (1 part Pharmacia Percoll mixed with an equal volume of MPD buffer) in 30
ml Corex siliconized glass centrifuge tubes. The two tubes were centrifuged in a swinging bucket rotor at 650 x g
for 60 minutes (4°C). The supernatants were then decanted, and each pellet was resuspended in 1 ml of MPD
buffer. Nuclear suspensions (approximately 3 ml total) were transferred to a single 15 ml polypropylene centrifuge
tube. 20% SDS was added to a final concentration of 2%, and the tube's contents were mixed by gentle inversion.
The resulting nuclear lysate was incubated in a water bath at 60°C for 10 minutes. The lysate was cooled to room
temperature, 5 M sodium perchlorate was added to a final concentration of 1 M, and the contents of the tube were
mixed. The tube was spun in a swinging bucket rotor at 400 x g for 20 minutes (20°C). The supernatant
(approximately 6 ml) then was transferred to a sterile polypropylene tube using a 1000 ml plastic pipette tip from
which the bottom 1/3 had been cut off. All subsequent transfers of solutions containing DNA were performed using
such modified pipet tips to minimize shearing of the DNA. The supernatant was mixed with an equal volume of
phenol/chloroform/isoamyl alcohol (25:24:1). Mixing of organic and aqueous phases was done using a test tube
rocker at 18 cycles/min to minimize shearing of DNA. The mixture was centrifuged at 3000 x g in a swinging
bucket rotor for 10 min at 4°C. The upper aqueous phase containing nucleic acids was placed into a new sterile
polypropylene tube, and a second extraction was performed. The aqueous phase was then dialyzed into TE at
4°C. After dialysis, DNase-free RNase T1 (Boerhinger Mannheim) and RNase A (Sigma) were added to final
concentrations of 50 units/ml and 50 µg/ml,
No navigation control above? Click here!