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Glyoxal RNA Gel Notes: Wear washed and rinsed gloves. Use a fresh box of pipette tips and a fresh box of tubes (label
 

Summary: Glyoxal RNA Gel
Notes: Wear washed and rinsed gloves. Use a fresh box of pipette tips and a fresh box of tubes (label
"RNA use only"). DEPC-treat solutions: 10 L dH2O; 2 L 10 mM NaPi, pH 7.0. Add 0.01% DEPC
(100 l/L), incubate at 37C for >2 hours, or overnight at room temperature, and autoclave.
Treat electrophoresis apparatus:
1. Wash apparatus with mild detergent and rinse thoroughly with dH2O.
2. Set up gel apparatus with pump and tubing. Pump 3% H2O2 through apparatus for 10 minutes
(with comb and dams completely submerged). Check that pump is working. Pour out H2O2.
3. Pump DEPC-treated dH2O through apparatus for 10 minutes (comb and dams submerged). Pour
out dH2O. Repeat rinse at least twice. Small amounts of residual H2O2 will degrade RNA.
Pour and run gel:
1. Pour 0.8-1.2% agarose gel in DEPC treated 10 mM NaPi, pH 7.0. Do not use ethidium bromide in
gel. Treat agarose with ~10 mM sodium iodoacetate (0.20 g for 100 ml gel). Add sodium
iodoacetate after melting and cooling agarose to ~70C (too hot to hold). Pour gel when flask is at
50C (can be held). Completely submerge gel in DEPC treated 10 mM NaPi, pH 7.0.
2. Combine up to 10 g RNA in 3 l with 15 l glyoxal/DMSO (G/D) solution (stored at -70C).
3. Incubate at 50C for 1 hour. Chill on ice. Spin 5 seconds.
4. Add 2 l of loading solution, mix completely, and load all (20 l) of sample.
5. Run gel at ~40-50 V (maximum) until dye is ~75% down gel. Recirculate buffer continuously.
6. Optional: stain for 1 hour with 1 g/ml EtBr in 0.1 M ammonium acetate (250 mls dH2O + 20 g

  

Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida

 

Collections: Biology and Medicine