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
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|
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)
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| 12.5 ml
| 10 x running buffer
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| 102.5 ml
| DDW
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| 6 ml
| 35 % formaldehyde (check that it is a fresh batch that doesn't contain precipitates)
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|
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.
RNA sample buffer (prepared fresh from frozen stocks):
For 500ul:
50ul 10 x running buffer
250ul deionised formamide
90ul formaldehyde
108ul water (DEPC-treated)
2ul ethidium bromide (stock concentration, 10 mg/ml)
Heat the samples at 65糃 for 5 minutes then to each add 3ul of RNA loading buffer. This consists of 0.25% bromophenol blue, 0.25% xylene cyanol in 20% Ficoll in DEPC-treated water.
5. Running the gel
Fill tank with 1 X MOPS (prepare 1X with DDW straight from the milliQ). Early protocols added formaldehyde to the running buffer but this is unnecessary. Run the gel at between 100 to 200V for several hours, until the xylene cyanol dye front has migrated 3 to 4 cm into the gel and the bromophenol blue is about 2/3rds down the gel. Circulate the buffer from end to end every half an hour, especially if running the gel at 200V. When the RNA has run an appropriate distance, photograph the gel including a ruler aligned with the wells. Pre-soak the gel in 20 x SSC while setting up the transfer (about 15 minutes).
B. Northern transfer
1. Setting up the transfer
The physical set-up for Northern transfer is identical to that for Southern transfer (see above) except that 20 x SSC is used as the transfer buffer (Note RNA is hydrolysed in strongly alkaline solutions within seconds!) and the membrane we use is GeneScreen Plus. Prewet membrane in DDW the 20 x SSC.
2. Post-transfer handling of the membrane
After overnight transfer, the position and orientation of the wells is marked on the membrane (#1 etc.) and it is rinsed gently in 2 x SSC for 5 minutes before being air dried in the fume hood. The membrane is baked at 80糃 for 2 hours and is then ready for pre-hybridisation and hybridisation.
3. Hybridize in Aqua hyb. with 0.1% SDS, 100ug/ml herring testes DNA (see Reagents).
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