Southern Gel/Blot Protocol

 

Southern Gel/Blot Protocol


Alkaline Capillary blotting to charged nylon (Hybond N+)

materials:

  • 1X TBE (0.089 M Tris base, 0.089 M Boric acid, 0.0027 M EDTA), can make 20X stock
  • Hybond N+ (Amersham Pharmacia Biotech)
  • 0.25 M HCl -- 21.55 ml 11.6N per 1000 ml 0.25 N
  • 0.4 M NaOH -- 16 g per 1000 ml for soaking, & 32 g per 2000 ml for transfer.
  • 2X SSC


1. Prepare a 1% agarose gel in 1X TBE and run well-resuspended, restriction-digested genomic DNA samples, 10-25 micrograms per lane at 20-30 volts overnight with copy standard controls and size markers. Ethidium bromide stain the gel after running, and photograph with rulers.

2. Rinse gel in distilled water, 30 seconds.

3. Place in 0.25 M HCl to partially depurinate DNA fragments and get strand cleavage to reduce length. Agitate slowly 30' at room temperature.

4. Pour off HCl and rinse gel with distilled water.

5. Pour off water and pour 1000 ml 0.4 M NaOH into dish with gel. Agitate slowly 20' at room temperature. This step denatures the DNA.

6. Set up downward transfer in 0.4 M NaOH:


7. Make a stack of paper towels 2-3 cm high in glass dish. Towel stack should be slightly wider than the gel.
8. Place 4 pieces of Whatman 3MM filter paper on top of the paper towels. Wet a fifth piece of 3MM filter paper and place on top.
9. Wet the Hybond N+ membrane in dH2O. Membrane can be slightly larger than the gel.
10. Place on top of filter paper. Remove bubbles by rolling a glass pipet over the surface of the membrane.
11. Place gel on the membrane and carefully remove bubbles. No part of the gel should extend over the membrane.
12. Place plastic wrap around gel to prevent wicking.
13. Soak 3 pieces of 3MM paper cut to the same size as gel in transfer buffer and place on top of gel.
14. Cut 2 larger pieces of 3MM for wicks, and soak in transfer buffer. Link from buffer reservoir to top of stack to form a bridge between the gel and the reservoir.
15. Place a light plastic cover over the top to reduce evaporation. Leave for 1-4 hours (Tim says 4 hr/ Red book says 1 hr).


16. Remove paper towels and filter paper and invert gel/membrane stack. Mark the wells of the gel and the orientation on the membrane.
17. Rinse membrane in 2 X SSC, rubbing lightly to remove agarose particles.
18. Place on 3MM sheet and allow to dry.
19. No baking or cross-linking is required for alkaline transfer to charged nylon.


Source is Current Protocols in Molecular Biology, 2.9.7, recommended by Tim Bender/Rajewsky lab.