4.12.1. Agarose /formaldehyde gel electrophoresis

What follows is a standard protocol for denaturing gels suitable for linear separation of RNA strands:

  1. Be RNase free!! Use gel apparatus designated for RNA. Wipe apparatus with “RNaseAway” and rinse thoroughly with RNAse-free water.
  2. Prepare 100 ml of 1% agarose/formaldehyde gel:
    1. Dissolve 1 g agarose in 72 ml DEPC-treated water in a 250 ml glass flask.
    2. Cool to 60oC in a water bath. 
    3. Add 10 ml of 5X MOPS running buffer (200 mM MOPS buffer, 50 mM Sodium acetate, 20 mM EDTA, pH 7.0) and 18 ml of 37% formaldehyde.
    Precautions: Formaldehyde vapours are toxic. Prepare the gel in a fume hood.
  3. Pour the gel to the gel tank and allow it to set.
  4. Add sufficient 1X MOP running buffer to fill the tank in order to cover the gel and remove the comb carefully.
  5. To prepare samples for gel electrophoresis, mix:
    11 ml of each RNA sample (0.5-1 mg/ml),
    5 ml 5X MOPS running buffer,
    9 ml 37% formaldehyde,
    25 ml of 50% formamide.
  6. Heat the sample at 65oC for 15 min.
  7. Cool on ice for 2 min.
  8. Add 3 ml loading dye mix and 2 ml ethidium bromide (0.5 mg/ml).
  9. Run the gel immediately after loading samples.
  10. When the gel dye bands have separated and migrated at least 2-3cm into the gel, or as far as 2/3 the length of the gel, visualize under UV light and take picture.

The 28s and 18sribosomal RNA (rRNA) should appear as sharp bands on the gel with no apparent smearing from degradation.  The 28S rRNA band should be approximately twice as intense as the 18S.