Northern Blot Protocol

Use disposable plastic when possible to avoid RNase contamination. Isolate glassware specifically for RNA use. Wear gloves. Use DEPC water. Avoid keeping RNA at room temperature. Soak box, combs, etc. in 3% H2O2 for a few hours before using, allow to air dry completely. Be generally paranoid.

- Prepare formaldehyde-containing gel.
- Combine about 15 uL sample buffer with 5-10 uL RNA sample. Can use 5-20 ug RNA.
- Heat RNA samples/sample buffer 5 minutes at 65C before loading. It is not necessary to snap cool the samples after this heating.
- Run gel in 1X MOPS Buffer at 40-60V for medium size gel; allow 2+ hours.
- Visualize with long-wave UV light. Take a picture UV illuminator
- Fill casserole dish with 20X SSC. Place support for gel transfer in dish. Drape about 3 large whatman filters over the box so they wick up the SSC.
- Measure gel. Cut Hybond N+ or GeneScreen Nylon filter to the size of the gel. Allow it to presoak in water 30-60 minutes.
- Cut about 20 whatman filter papers to size of gel; also, about 10 cm worth of paper towels.
- Drip 20X SSC onto the wick. Flip the gel and place on wick (so smooth side is up).

- Place the hybond on the gel and smooth out, roll with a pipette. Make sure you get rid of air bubbles. Once the hybond has contacted the gel, do not move it. Clip a corner of the hybond for orientation.

- Stretch parafilm or plastic wrap to cover the edge of the hybond.
- Place dry filter paper on top of the hybond. Stack paper towels on these. Weight the entire pile down with 300-500 g weight (depends on size of gel).
- Transfer overnight.
- Place the gel in the UV crosslinker. Turn on, select "optimal crosslink" and hit start. The light should come on and the timer should count down.
- Wash in 5X SSC/0.1% SDS at room temperature several times to remove formaldehyde. Visualize with UV light, use a membrane marker to mark 28S and 18S RNA.
- Place hybond in tupperware and incubate in 5X SSC/0.1% SDS for one hour at 65C in a shaking water bath to adequately wet the membrane.
- Boil 50 uL salmon sperm DNA (10 mg/mL) for ten minutes, quench on ice 2 minutes. Thaw prehyb solution. Add Salmon Sperm DNA to prehyb. Place membrane in seal-a-meal bag. Add prehyb solution.
- Seal bag. Again, incubate at 65C. Prehyb one hour to all day long.
- Boil DNA probe plus an additional 50 uL salmon sperm DNA for 10 minutes in a screw-top eppendorf. Briefly spin to pellet fluid. Add 500 uL prehyb solution, and then add to the bag. Avoid spotting directly onto the nylon membrane; instead, tilt bag and add to fluid.
- Work out the air bubbles and reseal the bag. Hybridize overnight at 65C.
- Drain bag onto absorbant napkin. Remove hybond, wash a few times in 5X SSC/0.1% SDS to get rid of the bulk of the radioactivity. Empty sink. With water running, pour directly into the drain. Repeat as necessary. Wash successively in increasingly stringent dilutions of SSC. Start with a 2X SSC/0.1% stock and serially halve the strength, i.e., 2X -> 1X -> 0.5X -> 0.25X -> 0.125X.
- Blot quickly on whatman paper, but do not allow the membrane to dry at any point.
- Cover with saran wrap, smooth surface. Fold the edges of the Saran Wrap to capture any excess fluid.
- Tape the membrane onto a scrap piece of film, apply bits of glow-in-the-dark tape to orient the film.
- In dark room, position BioMax film over the membrane. Add intensifying screen. Close casette.
- Place at -80C overnight. In morning, develop film.
- Position film above the membrane. Draw dots for the 18S and 28S bands to help in orientation

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