Southern Blotting
1. Photograph the gel with a ruler along the left-hand side.
2. Soak gel for 15 min in 0.25 N HCl, with gentle shaking.
3. Pour off and soak gel for 1 x 30 min in Southern Denaturing solution with gentle
shaking.
4. Soak gel for 1 x 30 min in Neutralization solution with gentle shaking.
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 neutralisation buffer 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
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 3 cm.
16. Place a perspex sheet on top of the towels and a weight, approximately 500g.
17. Blot the gel overnight.
18. The following day, dismantle the blot, place the filter DNA-side up on 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.
21. Fix DNA to membrane by UV-crosslinking (in the crosslinker,setting 120), or by
baking at 80oC (under vacuum for nitrocellulose) for 2 hours (**do not use UV
with nitrocellulose membranes)
Southern Denaturing Buffer
0.4 N NaOH
0.6 M NaCl
Neutralization Buffer
1.5 M NaCl
0.5 M Tris-HCl pH 7.6
20X SSPE
3.6 M NaCl
840 g
0.2 M NaH2PO4.H20 110.4 g
0.02 M EDTA
33 g tetrasodium salt
adjust pH to 7.4 with 10 N NaOH
final volume = 4 l