Department of Biological Science, Stanford University, Stanford, CA 94305-5020, USA
Preparation of etiolated seedlings
a. Surface sterilize seed at room temperature with 5% solution of commercial bleach for 30 minutes. Use 400-600 g of seeds.
b. Rinse 3 times with sterile water and imbibie for two nights in sterile water with 1 tablesppon for fungicide ("Cooke Fungicide").
c. When seeds are imbibed, repeat the surface sterilization treatment, and then wash 3 times with sterile water.
d. Surface sterilize large, opaque plastic food storage boxes and sheets of "needle point" plastic templates with 10% bleach. The needle point template size are #7 for the bottom of the seed tray and #5 for the top; these sizes refer to the number of squares per inch.
e. Sow the sterilized rice seeds onto the size #7 bottom template, and then place the #5 template on top. String the two templates together with pipe cleaners. As the seeds germinate they will grow through the holes in the top plastic template. Set the template on about 1 cm supports (corks or a plastic rod-we use sections of fluorescent light fixtures) in a tray filled with water to just touch the level of the seeds. Cover the storage box with sterile, heavy duty aluminum foil to keep out light and keep humidity high. Identify seed source and note date on the tape tag on each box.
It is very important to exclude light, because a little light is sufficient to
cause greening and plasitc maturation; the seedlings will then contain more
chloroplast membrane which shows up as "green slime" contamination of the
preparation.
f. Grow seedlings for 6 to 8 days at about 25 degrees C. Shoots are best harvested when 2-3 cm tall, just before the leaves emerge through the coleoptile. If the correct amount of water was initially added, no additional watering should be necessary.
Preparation of mitochondria
All solutions should be prepared and ready to be used well n advance; chill all solutions to 0 to 4 degrees C so that each step proceeds with a minimum of time. Rotors should be pre-chilled, frozen solutions thawed, BSA and mercaptoethanol added to the HB (homogenization buffer), etc. We use sterile disposable plasticware when possible.
a. Before proceeding make sure the following items are available:
1. Chilled, steril 0.25 liter centrifuge bottles.
2. Chilled GSA rotor.
3. Add BSA (1 g/l) ad B-mercaptoethanol (3 ml/l) to 1 L HB.
4. Make 2 L of 10% bleach, chilled to 0-4 degrees C.
5. Chill 3 L sterile water.
6. Assemble funnels (large 10 cm plastic funnel is fitted with 1 layer miracloth, then 4 layers of cheesecloth; the whole assembly is then wrapped in aluminum foil and autoclaved as a unit), and sterile very fine (sable) oil paint brushes.
7. Weigh 1 liter plastic beaker and put it on ice.
8. Check availability of centrifuge, rotors and tubes.
9. Surface sterilize blender cup, chill to 0-4 degrees C.
10. Prepare 100 ul of 10 mg/ml DNase I.
11. Turn on the ultracentrifuge (vacuum), set temp to 4 degrees C.
b. Tissue preparation: Cut etiolated shoots that have emerged bove the plastic
template-work quickly and wear gloves for this and all subsequent operations.
Surface sterilize knife and/or razor blades before use. We use a fish fillet-
ting knife or new, single-edge rqzor blades to mow down the seedlings by
running the cutting tool firmly along the plastic template. Deposit shoots in
the weighted plastic beaker, kept in an ice bucket. Weight the bucket+shoots,
calculate and record the mass. We typically get 100 g of shoots from 100 g of
seed. Surface sterilize with 10% bleach for 10 min at 0 degrees C. Decant and
rinse 2-3 times with cold sterile water.
c. Homogenization: cut the shoots into smaller pieces (2 cm) using a large
scissors and transfer to the sterile blender cup or container. Homogenize with
several short pulses (3X5 s) in a Waring blender of a Polytron tissue
homogenizer (3X15 s) using approximately 2 ml HB/g tissue. Filter the
homogenate through the funnel, miracloth cheesecloth setup. Squeeze the
cheesecloth to obtain the maximum recovery of filtrate, but don't waste time
with the last few ml. Collect the filtrate in sterile, clear plastic 0.25 liter
contrifuge bottles.
d. Differential centrifugation: Quickly balance the bottles and centrifuge the
filtrate at 2,000 X g for 10 min (3,5000 rpm in GSA rotor). Transfer the
supernatant to a fresh sterile polypropylene (opaque plastic) centrifuge bottle
and centrifuge at 12,000 X g for 15 min. pour off and discard the supernatant
as well as any loose pellet on the surface of the firm mitochondrial pellet.
Resuspend the pellet with a minimal volume of HB and disperse well. It is moswt
effective to use a brush, working from the margin of the pellet to the center.
Avoid dislodging clumps, because these tend to be difficult to disperse in free
solution-thus keep the pellet dry and gently work buffer up with a brush to the
margin of the pellet. It is critical to completely disperse the pellet so that
the DNase will have access to any nuclear DNA which is present-clumps hinder
this accessibility.
e. DNase treatment: Resuspend the mitochondrial pellet in a final volume of 10
ml (per 4 trays, 400 g seeds) and add 0.1 ml of 1 M MgCl\2\ and 40 ul of 10
mg/ml DNase I. Incubate at 0 degrees C for 1 hr. Terminate digestion with 0.4
ml of 0.5 M EDTA. Note that the sucrose solutions are prepared with 10 mM EDTA
to insure that DNase I is inactive.
f. Sucrose denisty gradient centrifugation: Prepare a gradient in a 14X89 mm
polyclear unltracentrifuge tube (for SW41 rotor). Use 1.8 ml layers of 52%,
46%, 40%, 36% and 20% sucrose in HB.
Overlay the resuspended pellet, 3 to 3.5 ml per tube, and balance on an open
pan balance to within 10 mg. Keep gradients cold! Centrifuge in SW41 rotor for
50 min at 30,000 rpm (154,000 X g). Do not oberload the gradient; use 6
gradients for 400 g of starting material.
Remove upper layers of sucrose and carefully clear the interfaces to remove contaminating lipid or organelle fractions.Carefully collect the 40/46 and 46/52% sucrose interfaces with a sterile pasteur pipet. These fractions are diluted slowly over 10 min to 4 times their original volume with HB and then centrifuged at 9,500 rpm intheSS34 rotor (11,000 X g) for 10min. We use a sterile polypropylene tube (Falcon 2095). The pellet is resuspended in an appropriate volume (1 ml) of HB and transferred to a sterile microfuge tube. The supernatant is removed together with the surface material ('green' or 'yellow slime') leaving the buff-colored, purified mitochondrial pellet.
g. DNA extractions: Resuspend the pellet in 0.5 ml of 50 mM Tris-HC1, 10 mM
EDTA at pH 7.5. Solubilize the mitochondria with 50 ul of 10% Sarkosyl and add
200 ug (50 ul of 40 mg/ml) of proteinase K; the suspension should clear
immediately. Incubate at 37 degrees C for 30 min. A small amoutn (5 ul of 10
mg/ml) of heat-treated RNase may be added at this point if desired. After the
digestion(s), extract with an equal volume of TE-equilibated phenol and bck
extract the phenol with 0.5 volume of TE or sterile water. Pool the aqueous
phases and extract with chloroform (equal volume). Precipitate with a 0.1 volum
of 3 M sodium acetate and 3 volumes of ethanol at -80 degrees C for several
hours to overnight. Nucleic acids may be collected by centrifugation in a
microfuge for 10 min, washed with 70% ethanol and dried briefly in a speedy-
vac. Quantitation of the nucleic acid yield is done spectrophotometrically. To
check that most of the sample is DNA, prepare a 0.7% agarose gel and load 1 ug
of nuclei acid in a 5 mm well. Electrophoresis for 1 hr at 50 milliamps should
separate RNA from the DNA. If the sample is primarily high molecular weight
DNA, you will see a single band near the origin after staining with ethidium
bromide. RNA will have moved about 4-5 cm and be a smeary band near the dye
front.
An alternative to the phenol extraction for DNA purification, in centrifugation
on a CsCl gradient.The digested lysate may be diluted to the desired volume
(determined by the size of the ultracentrifuge tube) and made 1.7 g/ml with
CsCl. The solution is centrifuged as 50,000 rpm in the VTi80, and then
fractionated by dripping from the bottom. The nucleic acid-containing fractions
are analyzed by electrophoresis, and the desired fractions are dizlyzed and
nucleic acids collected by ethanol precipitation.
Solutions:
1) HB (homogenization buffer). 0.4 M mannitol, 1 mM EDTa, 0.05% cysteine, and 10 mM TES-Na+, pH 7.2. Autoclave and store at 4 degrees C. Supplement with 0.1% BSA and 0.03% beta-mercaptoethanol before use. Add BSA to the surface and allow to dissolve, do not stir.
2) Sucrose solutions are percent (w/v) prepared in 10 mM TES-Na+ pH 7.2, 10 mM EDTA and autoclaved.
3) 50 mM Tris-HCl, 10 mM EDTA (autoclave).