4.12.1. Agarose /formaldehyde gel electrophoresis
What follows is a standard protocol for denaturing gels suitable for linear separation of RNA strands:
- Be RNase free!! Use gel apparatus designated for RNA. Wipe apparatus with “RNaseAway” and rinse thoroughly with RNAse-free water.
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.
- Pour the gel to the gel tank and allow it to set.
- Add sufficient 1X MOP running buffer to fill the tank in order to cover the gel and remove the comb carefully.
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.
- Heat the sample at 65oC for 15 min.
- Cool on ice for 2 min.
- Add 3 ml loading dye mix and 2 ml ethidium bromide (0.5 mg/ml).
- Run the gel immediately after loading samples.
- 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.