Northern Blot

  1. Clean the gel box, gel mold and combs and soak them in 1%SDS for at least 2 hours, rinse with water.
  2. Prepare 10X MOPS and 20X SSC. 
    MOPS: 200 ml. 16.76 g MOPS, 2.72 g NaAc, 4 ml 0.5M EDTA. Keep the solution in dark, @4oC. 
    20X SSC: 1 l. 175.3 g NaCl, 88.2 g NaCitrate. Adjust pH to 7.4 with HCl.
  3. Prepare agarose gel and running buffer.
    Agarose gel: 200 ml (0.8%). 1.6 g agarose, 20 ml MOPS, 170.2 ml water. Boil to resuspend. Just before pouring add 0.8 ml formaldehyde (37%).
    Running buffer: 1 l. 100 ml 10X MOPS, 16.3 ml 37% formaldehyde, 83.7 ml DEPC treated water.
  4. Prepare RNA samples: bring all the samples to the same volume (up to 20 μl), add RNA loading buffer containing EtBr, denature samples @95oC/5min, cool on ice. RNA marker 0.5-10 kb ladder, Invitrogen SKU# 15623-200.
  5. Run gel in the fume hood (50-70 mA).
  6. Rinse gel 3 times for 5 min in DEPC treated water to remove formaldehyde.
  7. Photograph gel with a ruler beside it.
  8. Soak the gel for 20 min in 0.05 N NaOH to hydrolize RNA and improve efficiency of transfer (recommended if the gel is >1% or is >0.5 mm thick or the RNA to be analyzed is >2.5 kb).
  9. Cut the upper left corner of the gel, soak the gel in 10X SSC.
  10. Cut the membrane, soak it in DEPC water for 5 min, then in 10X SSC.
  11. Set up capillary blot with 10X SSC transfer buffer: 2 wet Watmann-gel -membrane - 2 wet Watmann -1 dry Watmann - paper towels – weight.
  12. Transfer ON.
  13. Cut the upper left corner of the membrane when it is facing down, mark the gel wells with a pencil, rinse the membrane briefly in 2X SSC to remove gel pieces.
  14. Dry the membrane on a dry Watmann for 30 min.
  15. Bake the membrane @ 80oC for 1-2 h.
  16. Prehybridize the membrane @68oC for 30 min using ExpressHyb hybridization solution (Clonetech; #636831).
  17. While prehybridizing, label the probe. When random priming, use GE Healthcare  Redi-to-go DNA labeling beads (27-9240-01). Use ≤50 ng of linearized probe resuspended in 45 μl of water, 5min/95oC, cool on ice and add to one ReadyPrime tube to reconstitute the labeling mix. Finally, add 5 μl of 32P dCTP. Labeling reaction 15-30 min.
  18. Hybridization- add denatured 32P-labeled probe to fresh pre-warmed hybridization buffer (ExpressHyb hybridization solution (Clonetech; #636831)) and substitute the prehybridization buffer with it.
  19. Hybridize 45 min-1 hour.
  20. Washings: 3X10 min/RT in  2X SSC/0.05% SDS.
  21. 2X20 min/50oC in 0.1% SSC/0.1% SDS.
  22. Wrap the membrane in saran wrap and expose to X ray film in a cassette with two amplifying screens at -80oC.
  23. Stripping: heat the sterile 0.5% SDS to boil.
  24. Place the blot in hot 0.5% SDS and incubate 10 min shaking frequently.
  25. Allow to cool down to RT.
  26. Remove the blot and air dry it. Store @ -20oC.