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 residual
           polyacrylamide 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 20 min.
 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 with water
       -- smooth one of the two cellophane sheets over the solid backing plate
       -- center the gel on the cellophane
       -- smooth the second sheet over gel
       -- 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%)                      Stacking Gel

30% acrylamide            4.7 mL                                        750 µL
 2% bis                         1.9 mL                                        300 µL
1 M Tris-HCl                5.0 mL (pH 9.0)                         320 µL (pH 6.8)
RNA solution                25-30 mg (volume=X µL)              -
0.1 M Tris pH 9.15      (730 - X) µL                                  -
H2O                                -                                              3.6 mL
10% APS*                   100 µL                                        40 µL
TEMED*                      10 µL                                          4 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.)
 

Running Buffer                           2x Sample Loading Buffer

Glycine           21.64 g                  50 mM Tris, pH 6.8       0.50 mL 1 M Tris, 6.8
Tris base          5.00 g                   2% SDS                        1.00 mL 20% SDS
SDS                1.50 g                   10% Glycerol                 1.25 mL 80% glycerol
H2O                1.50 L                  0.025% BPB                  0.25 mL 1% BPB
                                                    H2O                               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 volume).

 8. Check A260 of 1:1000, 1:5000 and 1:10,000 dilutions to quantitate.
 

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