A Detailed Procedure for RNase Activity Staining on SDS-PAGE
1. Remove glass plates that have been stored soaking in 3% hydrogen
peroxide and dry thoroughly with Kimwipes.
2. Assemble plates and seal with melted 1.5% agarose.
3. Prepare separating and stacking gels in the order listed
in the formula:
-- After pouring separating gel, slowly
drop Tris-saturated butanol (use 1M Tris pH 9.0 for this) to form
layer on the top
of the gel.
-- When the gel is polymerized, wash
off the butanol-saturated water with sterilized water and dry water and
thoroughly with filter paper.
-- After pouring stacking gel, enter
comb on its top on angle to avoid air bubbles.
-- When set, remove comb gently and
rinse wells thoroughly with running buffer.
4. Attach plates to running tank, add running buffer to
top and bottom tanks, and remove air bubbles from beneath the gel.
5. Load samples, connect power supply and run gel until
the front ion line reaches the bottom.
6. Remove plates from the tank, separate the gel from
the plates, and remove the stacking portion.
7. Wash the gel in 25% iso-propanol in 0.01 M Tris pH
7.0 for 2 x 10 min (to remove SDS from the gel).
8. Wash the gel in 2 µM ZnCl2/0.01 M Tris pH 7.0 for 2
x 10 min (to remove iso-propanol from the gel).
9. Incubate gel in 0.1 M Tris pH 7.0 at 51oC for 50 min.
10. Wash the gel in 0.01 M Tris pH 7.0, for 10 min.
11. Stain the gel in 0.2% toluidine blue O/0.01 M Tris pH 7.0
for 10 min.
12. Destain the gel in 0.01 M Tris pH 7.0 for 10 min and 2 x
13. Rinse the gel in 10% glycerol/0.01 M Tris pH 7.0 for 10 min.
14. Sandwich and dry the gel with cellophane.
-- cut two pieces of cellophane
to the size of the frame
-- wet the cellophane thoroughly
-- smooth one of the two cellophane
sheets over the solid backing plate
-- center the gel on the cellophane
-- smooth the second sheet over
-- clamp the frame over this sheet
using several binder clamps
-- set aside to dry overnight
-- remove dried gel from frame,
cut off excess cellophane, and store the gel flat.
To record the gels, we photograph them using a 35 mm camera mounted
above a white light box, and using Kodak "technical pan" film. Place
the gel on a glass plate on the light box, covering up the rest of the
light box surface with black paper to block the light around the gel.
Use of a yellow plastic filter underneath the gel improves definition.
We take a range of photographs at a shutter speed of 1/8 second and f-stops
of 5.6 to 11. Adjust if necessary for your equipment.
Separating Gel (11%)
1 M Tris-HCl
5.0 mL (pH 9.0)
320 µL (pH 6.8)
25-30 mg (volume=X µL)
0.1 M Tris pH 9.15 (730 - X) µL
* add just before pouring
(Adjust separating gel recipe as necessary according to the concentration
of your RNA sample. See protocol for preparing RNA below.)
2x Sample Loading Buffer
50 mM Tris, pH 6.8 0.50 mL 1 M Tris,
Tris base 5.00
1.00 mL 20% SDS
1.25 mL 80% glycerol
0.25 mL 1% BPB
bring to 10.00 mL
Preparing RNA to cast in RNase activity gels
1. Dissolve 100 mg/ml of torula yeast RNA (Sigma R-6625) in 1
M Tris pH 9.0 (100 ml is a convenient volume to make).
2. Phenol extract RNA solution, spinning 10 minutes at 7000 rpm.
3. Chloroform extract RNA twice, spinning 10 minutes at 7000 rpm.
4. Precipitate with 1/10 volume of 3 M NaOAC and two volumes of
EtOH. Spin 10 minutes at 12,500 rpm.
5. Wash pellet once with 70% EtOH.
6. Dry pellet in lyophilizer or speed-vac, with tube covered with
Parafilm that has holes poked in it.
7. Dissolve pellet in 0.1 M Tris pH 9.0 (about half the starting
8. Check A260 of 1:1000, 1:5000 and 1:10,000 dilutions to quantitate.
to the Green Lab Homepage.
Questions or comments? Mail