Northern Blotting
1. Photograph gel with ruler along left-hand side.
2. Cut off markers and bottom left-hand corner of gel.
3. Place gel in tray and add tepid DEPC-treated water to cover gel.
4. Gently shake for 15 min.
5. Set up tray with perspex sheet on top and make a wick with 3MM paper.
6. Into tray place 20X SSPE to form a reservoir of buffer.
7. Measure gel and cut 10 pieces of 3MM paper to gel size.
8. Carefully cut Hybond N (between protective sheets) to gel size.
9. Remove water from tray and carefully invert gel and place on wick.
10. Seal around the edges of the gel with either used X-ray film or Saran wrap, to
avoid any short-circuiting of the blot.
11. Cover the remainder of the reservoir with Saran wrap to avoid loss due to
evaporation.
12. Mark the Hybond N with the date and your initials (in pencil) and carefully place
(writing side facing gel) on top of the gel.
13.Wet 5 pieces of the cut 3MM paper with 20X SSPE and place on top of the
membrane.
14.Add the remaining 5 pieces of dry 3MM on top.
15. Stack brown towels on top of the 3MM paper to a height of approximately 3cm.
16. Place a perspex sheet on top of the towels and a weight, approximately 500g.
17. Blot overnight.
18. The following day, dismantle the blot, place the filter RNA-side up on a paper
towel and allow to air-dry for 1 hour.
19. Discard the gel in the biohazard bag.
20. After 1 hour wrap the filter in Saran wrap and UV-crosslink on the UV
transilluminator for 6 min (RNA-side down).
21. Store filter safely until required.