RNA formaldehyde gels
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Procedure
Prepare gel box:
- Clean gel box and combs etc with detergent, then dionized water.
- Then incubate the gel box and components in 10% H2O2 (hydrogen peroxide, stored in cold room) for 30 minutes.
- Rinse box thoroughly with DEPC water.
For making gel and preparing samples:
- Your should pour (and ideally run) the gel in the hood, since the gel contains formaldehyde. [see protocol from Maniatis (original edition)—attached.]
Running the gel:
Just before loading the samples:
- Heat your samples (see step 3 Maniatis protocol) for 15 minutes at 65 or 70 C and then put immediately on ice. The add loading buffer (see step 4 Maniatis).
- ALSO, pre-run the gel at 50 V for 10 minutes
- Load samples.
- For mini gels, run at 30V for 4-5 hours. (for best results place the gel on a bed of ice to keep it cold)
- Stain in EtBr 1 hour to overnight ( 5 µl of 10 mg/ml solution per 50 ml DEPC water)
- Destain in DEPC water for 1-2 hours (change water 2x during this time)
- Take picture (Karla's lab has a good digital documentation system)