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Harvest cells when they are about 80-85% confluent. Stationary phase cells have decreased RNA yield.
Wash cells with warm PBS and scrape them off in 7 ml cold GITC buffer.
Pour GITC/cell mix into (14x95 mm) polyallomer tubes with 4 ml CsCl buffer.
Balance and top off tubes with more GITC buffer.
Spin tubes on the SW40Ti rotor for 20 hr at 32,000 rpm (18°C).
Pour off liquid and keep tubes inverted because the RNA pellets on the bottom. (DNA can be recovered from the CsCl layer.)
Cut off the tube bottoms (keep inverted) and resuspend the pellet in 100 µl 0.3 M NaOAc (pH 6) buffer. Transfer to a screw-cap 1.5 ml Eppi. Wash tube bottom twice more with 100 µl buffer each time.
Add 750 µl 100% EtOH and let precipitate overnight at -70°C.
Spin down at 4°C for 12 min, dry in the TC hood, and resuspend with DEPC water.
Let resuspended RNA sit on ice for at least 30 min before quantitation.
Pipet up and down several times before taking 2 µl into 998 µl water.
Read at 260 nm.
RNA concentration (µg/µl) = OD x 500 x 0.04 µg/µl = OD x 20
Boil 2 g agarose in 144 ml water (check weight to account for evaporation)
Add 20 ml 10X MOPS and let cool on stir plate to about 56°C.
Add 36 ml 37% formaldehyde.
Pour into Hoefer appartatus when gel solution is below 50°C.
Running buffer: dilute 100 ml 10X MOPS to one liter.
To 0.5 ml Eppis, add: a) 15 µg RNA (approxiamately 4.5 µl) b) 14.5 µl sample buffer c) Adjust with DEPC water so that final volumes are equal d) Heat for 5 min at 68°C, then chill on ice for 5 min. e) Add 2 µl loading buffer and 1 µl 0.4 mg/ml EtBr.
For the ladder lanes, use 3 µg of 1 kb ladder and treat the same way as with the samples.
Running the gel
For the Hoefer, run between 70-85 V for about 3 hr or to a predetermined length.
Recirculate the buffer with a peristaltic pump.
As the gel is running, cut membrane, paper towels, etc.
At the end, take a picture (f8, 4s) of the gel using a fluorescent ruler.
Fill reservoir with 10X SSPE. Lay a piece of chromatography paper across a glass plate. Allow the SSPE to wick up the paper (or just soak the paper wet).
Remove all bubbles between the paper and the plate by rolling a pipet across.
Lay gel top-side down and remove bubbles between gel and paper.
Surround the gel with Parafilm strips.
Soak the MagnaGraph membrane in ddH20 for 3 min and lay it on top of the gel.
Place 2 pieces of chromatography paper on top of membrane.
Stack at least 3 inches of cut-to-size brown paper towels on top of paper.
Place an oversized piece of plastic or glass on top of the towels. Weigh the whole set up down with about 400 gm (like water in tupperware).
Allow the RNA to transfer overnight.
Bake membrane in 80°C vacuum oven for 20 min.
UV crosslink (on optimal setting of Fisher Crosslinker) the membrane (face side up).
Store between 2 pieces of chromatography paper in a plastic bag.
Labelling the probe
Make a 10 ng/µl dilution of the probe.
Add 10 µl (100 ng) of probe to a 0.5 ml screw-cap eppendorf tube. Adjust volume to 28 µl with water. (ie, 7 µl of probe needs 21 µl H2 O.)
Boil for 5 min and then chill on ice for 5 min.
Briefly spin up/down in the microfuge.
Following the protocol from the Amersham Multiprime Kit (RPN.1601):
Add 10 µl of Buffer (solution 1, blue cap vial)
Add 5 µl of Primer (solution 2, black cap vial)
Add 5 µl of [a-32P]dCTP (3000 Ci/mmol)
Add 2 µl of Enzyme (solution 3, Klenow fragment) Final volume at this point should be 50 µl.
Incubate for 1 hr at 37°C or overnight at room temperature.
Prehybing the blot
For hybridization in a tube, about 3-5 ml of buffer is sufficient. If a bag is used, it's about 0.2 ml of buffer per square cm of membrane. It is critical that the mem- brane be wet thoroughly.
Boil 200 µg/ml of sheared salmon sperm DNA in a 50 ml conical tube for 5 min and chill on ice for 5 min. (If it's a total of 10 ml, use 2 mg salmon sperm.)
Add the hybridization buffer (5 ml) and the formamide (5 ml) to the salmon sperm.
Put the membrane in a tube and wet it with this mixture.
Incubate at 42°C for at least 30 min.
Check the incorporation of [32P]dCTP into the probe first.
Spot 1 µl of reaction mixture onto the center of a small piece of DE81 paper that is placed into the bottom of a 7 ml scintillation vial. Do this twice (i.e., into two vials. One will be washed; the other will be the control.)
Let the spot dry (about 2 min).
Fill the wash vial with 5 ml of 0.5 M Na2HPO4and let it sit for 3 min. Then aspirate off the wash. Do this twice more.
Add 5 ml of scintillation fluid and count.
% incorporation = (cpm of washed/cpm of unwashed) x 100. Anything over 10% is usually usable. Using the kit, it's usually 40%.
The specific activity (S.A.) of the probe is also important. S.A. = [(cpm of washed) x (volume of labelling reaction)]/µg amount of DNA If S.A. is over 1x108, the probe is OK.
Boil the probe for 5 min and chill it on ice for 5 min.
Up/down spin the tube to bring down the condensation on the lid.
Add it to the tube or bag.
Let it hybridize overnight (at least 16 hr) at 42°C.
Washing the blot
Add about 20 ml 2X SSPE/0.1% SDS to the tube. Mix and pour out. Be careful of loose drops.
Fill tube ≈ 1/3 -1/2 full with 2X SSPE/0.1% SDS and incubate at 56°C for 30 min.
Repeat with 1X SSPE/0.1% SDS and 0.1X SSPE/0.1% SDS until background counts are below 1000 cpm. Keep washing with 0.1X SSPE/0.1% SDS if the counts are high. Washes can go longer than 20 min. The stringency of the washes depends on the nature of the probe.
Wrap the blot with Saran wrap making sure there are no wrinkles.
Expose to autoradiography film overnight or to a Molecular Imager screen for 1 hr. Adjust exposure time accordingly. The time on the screen is roughly one-tenth to what it would be on film.
GITC buffer (200 ml):
94.53 g Guanidine isothiocyanate (4 M final)
1.67 ml 3 M NaOAc, pH 6
Bring volume to 200 ml
Add 1.67 ml ß-mercaptoethanol
Store a 4°C and away from light
CsCl buffer (100 ml):
95.97 g CsCl (5.7 M final)
0.83 ml 3 M NaOAc, pH 6
Bring volume to 100 ml
Sterile filter with 0.8 µm
(This is optional.)
0.4 M 3-[N-Morpholino] propanesulfonic acid, pH 7.0
0.1 M Sodium acetate
10 mM EDTA (pH 8.0)
Prepare in DEPC water. OK if solution turns yellow.
Autoclave to sterilize.
Add DEPC to ddH2O at 1:1000 dilution
Let it sit overnight at room temp (destroys RNase)