Northern Blot analysis.

Combination of protocols from Current Protocols in Molecular Biology and GeneScreen Plus Protocols

Prepare Gel

1. For standard mdm size gel, dissolve 1.5g agarose in 108 ml DEPC H₂O. Boil then cool to 60° C in a water bath.
2. Place flask in fume hood once at 60° C and add 27ml formaldehyde and 15ml 10X MOPS. Swirl to mix then pour gel and allow to set.
3. Keep gel tank in fume hood due to presence of formaldehyde. Pour running buffer (1X MOPS in DEPC H₂O) in sufficient to cover gel.

Preparing Sample and Running Gel

1. Adjust volume of RNA sample (10μg) to a total of 5.5m l with DEPC H₂O then add 2.5m l 10X MOPS, 4.5m l 12.3M formaldehyde (stock concentration), and 12.5m l formamide. **Prepare RNA marker at same time.
2. Mix by vortexing and centrifuge briefly. Incubate at 15min at 55° C.
3. Add 4m l formaldehyde loading buffer and 1ul 10mg/ml Ethidium bromide, vortex and quick spin to collect liquid and load into gel.
4. Run the gel at 5V/cm (mdm gel ~70V) until the bromophenol blue dye has migrrated halfway to 2/3 way down the gel. Usually takes ~3hrs. (Note people in our lab have run mdm gels up to 120V without problems thus far.)
5. Take a picture of the gel with a ruler on the side so can measure placement of markers.

Transfer of RNA

1. Soak gel in 5vol distilled DEPC H₂O for 5 min shaking. Repeat 4X.
2. Place GeneScreen membrane, cut to size, in DEPC H₂O for a few seconds to hydrate.
3. Soak membrane in 10X SSC for 15min
4. Set up capillary blot using 10X SSC as the transfer solution. Be sure to remove all bubbles between the layers. (BOTTOM -> 3 pieces whatman, gel (back side up, membrane (Use pen or needle to mark wells and what will be the top left corner of membrance), 3 pieces whatman, stacked paper town and weight -> TOP)
5. Transfer 16-24h.
6. Carefully remove filter paper without disturbing membrane. The lift membrcae away from gel with plastic forceps.
7. Rinse the membrace briefly in 2X SSC to remove residual agarose. Fix RNA to the membrace using UV Stratalinker.
8. Place the membrane RNA side up on filter paper to dry. Can rehydrate in 2XSSC

Hybridization of Probe

1. Prehybridize membrane in a minimum of volume at 42° C for 2-4h with agitation. (Pre-heat Prehyb solution before placing on membrane.)
2. Remove prehyb solution and add fresh pre-heated hybridization solution and denatured probe ~5E5dpm.
3. Hybridize O/N at 42° C with agitation.

Washes

1. Wash blot 1x 5min RT 2X SSC
2. Wash 3X 20min each 65° C w/ 0.2X SSC/0.1%SDS
3. Wash 1X 5min RT 2X SSC
4. Wrap in plastic wrap and place on phosphoimager or film.

Stripping Northern:

1. Pour boiling stripping solution onto membrane then keep at 80-90° C for 5min.
2. Pour off this solution. Can repeat as necessary. Check blot on phosphoimager to ensure successful removal of probe.

Stripping solution: 1% SDS, 0.1X SSC, 40mM Tris pH7.5 (can store up to 1yr at RT)

Solutions: