1. 0.2 M Sodium Phosphate, pH 6.8: Dissolve 6.9 g monobasic and 7.1 g
dibasic salts in DEPC-treated dH2O, pH should be 6.8. If not, adjust.
Bring up to 500 ml. Remove aliquots, then autoclave.
2. Gel Buffer and Running Buffer, 10 mM Sodium Phosphate pH 6.8: 150 ml
of 0.2M up to 3L, pH then autoclave.
3. Transfer Buffer: 20XSSC (3M NaCl, 0.3M Sodium Citrate) pH 7.0: 175.0 g NaCl and
88.0 g Na Citrate per liter. Autoclaved.
4. Glyoxal (Sigma, 40%, RNase free, 4'C): Deionize by mixing in 10% (w/v) of
a mixed bed resin with 6.9 M Glyoxal. Repeat three more times.
5. 0.1% DEPC treated water: Add 1 ml per liter of DEPC, stir O/N then
autoclave for 1 hour.
6. Loading dye: 100 ul of 50% RNase-free Sucrose and 25 ul of 0.5% (w/v)
bromophenol blue and xylene cyanol in DEPC treated dH2O.
7. Denaturation Mixture: 72.5 ul Glyoxal and 25 ul of 0.2 M Sodium Phosphate.
Use 3.9 ul per sample.
Note: final concentration's=1M Glyoxal and 10 mM NaPO4.
1. Treat gel box and comb with 0.1% DEPC water. Thaw RNA on ice.
2. 1.0 % Agarose gel: dissolve 2.5 g Agarose in 250 ml of 10. mM Sodium
Phosphate for small HSI gel tray (3.5g and 350 ml for large HSI tray).
3. Pour gel. Let harden for 30-45 minutes.
4. Redissolve RNA for 5 minutes at 65'C. Spin down then remove 20 ug to a
new, labeled tube. Bring up to 16 ul with DEPC-treated H2O.
5. Add 3.9 ul of denaturant mixture to each sample.
Note: Include an RNA molecular weight marker as well.
6. Denature for 10 minutes at 65'C. Ice Until ready to load, spin down.
7. Make loading dye: 100 ul of 50% RNase-free Sucrose and 25 ul 0.5% dye.
8. Add 2.5 ul of loading dye to each sample, on ice. Vortex then spin down.
9. Add samples ASAP, while still on ice.
10. Run samples into gel at 150V, then turn down to 100V for 4-5 hours
with stir plate in place.