A. Formaldehyde agarose gel electrophoresis
Note: Formaldehyde vapours are toxic and casting this gel should be performed in a fume hood. The gel tank must be covered when in use.
1. Preparation of equipment and reagents
Soak the gel tanks, combs etc in 0.2 M NaOH for 15 minutes to destroy any contaminating RNases before rinsing in milliQ water. It is not necessary to use DEPC treated water.
2. Make up 10 x Northern running buffer (containing MOPS):
| Final conc: | For 500 ml: | |
| 0.2 M MOPS | 20.93 g | |
| 10 mM EDTA | 10 ml of 0.5 M | |
| 50 mM Na Acetate | 8.33 ml of 3 M | |
The buffer needs to be brought to a pH of 7.0. For 500 ml of 10 X MOPS this means add approx 2 g of NaOH. Make up to 500 ml in DEPC-treated water [To DEPC-treat solutions, add 0.1% DEPC to the solution in a bottle which can be autoclaved. Mix the solution well and allow it to stand, with the cap tightly closed, for at least 30 minutes. Then loosen the cap and autoclave. This must be done in a fume hood as DEPC is very toxic. Note also DEPC is inactivated by water. Allow the DEPC stock solution bottle to warm to RT before opening to avoid condensation. Parafiln shut].
Autoclaving of the MOPS buffer is not necessary and turns the solution yellow. Store at 4.
3. Cast the gel
Cast a 14 cm, 0.7-1.0% agarose gel, which requires at least 100ml of agarose solution. Note: ethidium bromide is not added to the gel but rather to the sample buffer (see below). A thin, low percentage agarose (0.6-0.7%) gel is critical for good transfer of large MW RNA's (>4-5kb).
| For 125 ml: | |
| 1.25 g | agarose (1% gel) |
| 12.5 ml | 10 x running buffer |
| 102.5 ml | DDW |
| 6 ml | 35 % formaldehyde (check that it is a fresh batch that doesn't contain precipitates) |
Dissolve the agarose in the microwave, let the solution cool to less than 60糃, then add the formaldheyde and cast the gel.
4. Prepare RNA samples for loading
RNA is stored at -80糃 as an ethanol precipitate. Determine the amount of RNA to be loaded in each well (e.g. 2ug of poly A(+) RNA). Based on your RNA yield estimations, precipitate the appropriate amount of RNA ethanol solution for at least 15 minutes at 13,000g at 4? Remove all supernatant and resuspend each sample in 12ul of sample buffer.
