Southern Blot
(Selker Lab)
Digest 1 g of genomic DNA overnight (or 5 ul of 50 ul miniprep).
Run digested DNA out on a 0.8% to 1.0% Agarose gel (1X TBE).
Take a picture and mark the standard ladder by poking holes with Pasteur pipet.
Transfer gel into a Pyrex backing dish.
Depurinate in 0.2 M HCl for 10 to 20 min. (until the blue dyes change to yellow).
Denature with 0.4 M NaOH for 10 to 20 min (till the yellow changes back to blue).
Set up transfer rig (glass plate with filter paper wick) in baking dish filled ¾ with
0.4 M NaOH.
8. Cut and label (with pencil) Magnaprobe (Zetabind, or other) membrane.
9. Wet membrane in H20, 2xSSC or 0.4 M NaOH.
10. Place denatured gel on transfer rig. Block sides of gel with plastic strips (to avoid
transfer bypassing the membrane).
11. Place Zetabind membrane on gel. Roll out any bubbles with glass pipet being
careful not to shift membrane.
12. Put filter paper on top of membrane.
13. Add stacks of paper towels.
14. Transfer overnight (or at least 8 hrs.)
15. After transfer, remove towels and filter paper. Flip gel and membrane without
shifting them relative to each other. Mark the position of the standard ladder and
wells on the membrane with a pencil. (Or you can mark right through the wells of
the gel with a pencil without flipping it.)
16. Wash membrane briefly in 2 X SSC to remove any agarose.
17. Briefly air dry but do not dry out completely (This step can be left out).
18. Crosslink DNA to membrane in UV crosslinker (set at optimal crosslink).
19. Transfer membrane to hybridization bottle.
20. Pre-wash at 64 C for 20 min. with pre-wash solution. (This step can be left out.)
21. Prehyb at 64oC for 15 min. in SDS Prehyb solution
22. Add boiled probe to bottle. (See random hexamer probe protocol. Add 150 μl of
TE before boiling probe).
23. Incubate overnight in Techne Hybridization oven.
24. Remove probe (either keep in hot freezer or discard in liquid waste).
25. Wash 2 X 15 min. with Zetawash solution at 64oC.
26. Wash 2 X 20 min. with Zetawash solution at 64oC. (for less stringent wash, use
50oC oven)
27. Wrap membrane in Saran wrap and expose to film.
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7.
Stripping Membrane
1. Put membrane in pre-hyb bottle with room temperature 0.4 M NaOH.
2. Incubate for 20 min. (strictly) at 50oC.
3. Pour off NaOH and add cold Stripping II solution.
4. Incubate for 20 min. at 50oC.
Random Hexamer Probe Labeling
Random Hexanucleotides
10 X Reaction Buffer (included with the enzyme):
500 mM Tris-HCl, pH 7.5
100 mM MgCl2
10 mM DTT
0.5 mg/ml acetyated bovine serum albumin (BSA)
3.5 A260 units
10 μl
sdH20
to 100 μl
1. Denature the linearized DNA (in a volume of at least 10 μl) by heating for 10 min. at
95oC and then chill on ice.
2. To a microcentrifuge tube on ice, add the following:
i. 25 ng denatured DNA (digested)
ii. 3 μl dATP, dGTP, dTTP mixture (1:1:1)
iii. 2 μl reaction mixture (random hexamers in 10 X reaction buffer)
iv. 5 μl [a-32P] dCTP, 50 μCi, 3000 Ci/mmole, aqueous solution
3. Adjust volume to 19 μl with sdH20.
4. Add 1 μl exonuclease-free Klenow enzyme (2 U).
5. Incubate for 30 min. at 37oC.
6. Add 150 μl of TE. (This step can be left out.)
7. Denature probe by heating at 95oC for 5 min.
*probes for methylated regions can be generated by PCR from plasmids in separate
collection in under-counter freezer in 355 using T3 and T& primers.