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RESEARCH DIVISION Laboratory Manual

 


 

Northern Transfer Protocol

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.

  1. Make up 10 x Northern running buffer (500 ml containing MOPS):

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. Paraffin shut].

Autoclaving of the MOPS buffer is not necessary and turns the solution yellow. Store at 4C.

  1. 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 60C, then add the formaldehyde and cast the gel.

  1. Prepare RNA samples for loading

RNA is stored at -80C as an ethanol precipitate. Determine the amount of RNA to be loaded in each well (eg. 2g 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 12l of sample buffer.

RNA sample buffer (prepared fresh from frozen stocks):

For 500l:

- 50l 10 x running buffer
- 250l deionised formamide
- 90l formaldehyde
- 108l water (DEPC-treated)
- 2l ethidium bromide (stock concentration, 10 mg/ml)

Heat the samples at 65C 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.

  1. 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

Blotting

  1. Wet membrane (Genescreen Plus - Du Pont NEF 988 - much better than Hybond NC) in distilled water for a few seconds until fully hydrated.
  2. Soak membrane in 10xSSC buffer for 15 min.
  3. Use 10xSSC as the transfer solution. Transfer O/N.
  4. Rinse the membrane briefly in 2xSSC to remove residual agarose.
  5. Bake the membrane at 80C for 2 hrs.

Hybridization

  1. Prehybridize membrane in a minimum volume of prehybridization solution at 42C for 2-4 hrs.
Prehybridization buffer: 5X SSC
- 50% (w/v) deionized formamide
- 5X Denhardt's solution
- 1% SDS
- 10% Dextran Sulphate, Na salt (MW 500, 000)
  1. Add the probe into the prehybridization solution (or remove prehybridization solution, then add prehybridization solution and probe.)
  2. Hybridize O/N.

Washing

  1. Wash membrane in 200ml of 2xSSC at room temperature for 15 min.
  2. Wash membrane in 400 ml of 2xSSC, 2% SDS at 65C for 45 min twice.
  3. Wash membrane in 200 ml of 0.1xSSC, 1% SDS at 65C for 30 min twice.

Stripping DNA probes

  1. Boil membrane for 30 min in a solution of 10 mM Tris-HCl, pH7.5-8.0, 1 mM EDTA, 1% SDS.
Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au
David Bowtell PMCI October 1998