Alkaline Southern gel transfer

Alkaline Southern gel transfer

Wear gloves because of EtBr, HCl, and NaOH. Wear goggles to protect from UV light.

  1. Soak the gel in 500 ml of 0.25 M HCl (21ml concentrated HCl per L) for 10 to 15 min (usually 10 min) on a shaker (slow speed: 10-20 rpm) to depurinate the DNA. Rinse with distilled water briefly. Hold the gel(s) to the bottom of the baking dish with gloved hand while poring out the liquid.

  2. Soak the gel in twice 500 ml (at least twice volume of total gel volume) of 0.2 N NaOH 0.6 M NaCl for 15 min to denature the DNA. Rinse briefly in distilled H2O.

  3. Soak the gel in twice 500 ml (at least twice volume of total gel volume) of 0.5 N Tris (pH 7.5) 1.5 M NaCl for 15 min to nutralize the DNA. Do not rinse. Float nylon membrane on the top of distilled H2O to wet thoroughly and transfer in 25 mM NaHPO4. Soak the sponge and 3MM paper in in 25 mM NaHPO4. Do not touch the membrane with bare hand. Finger print left.

  4. Place the sponge in a glass dish and add in 25 mM NaHPO4 even with the top of the sponge.

  5. Place 2 3MM papers on the sponge, flood within 25 mM NaHPO4, add gel (upside down), make a saran wrap mask, flood, add the membrane (numbered side up), squeeze out all bubbles, flood, add 2 wet and 3 dry 3MM papers, add 1 to 2 cm of white paper towels, and top off with a tall stack of paper towel with a 100-1000 g weight on top.
  6. Check the level of the in 25 mM NaHPO4 periodically (every 4 hr so) and keep it even with the top of the sponge. Allow to transfer overnight (be sure to refill in 25 mM NaHPO4 before leaving for evening).
  7. The next morning, remove top paper towel stack, mark the wells with a sharp lead pencil and discard gel.
  8. Rinse the membrane in 2X SSC. Bake the membrane between two sheets of dry 3MM papers for 3hr at 90 C in oven to immobilize the DNA.
  9. If the membrane is to be stored, seal it in a plastic bag between sheets of dry 3MM paper and store at 4 C.
0.2 N NaOH 0.6 M NaCl 
	Conc.		1 L	2 L 

	0.2 N NaOH	 8 g	16 g
	0.6 M NaCl	35 g	70 g
0.5 M Tris (pH 7.5) 1.5 M NaCl 
	Conc.		1 L	2 L


	0.5 M Tris	63.5 g	127   g	Tris-HCl
			11.8 g	 23.6 g	Tris-Base
	1.5 N NaCl	87.7 g	175   g	NaCl
25 mM NaHPO4 
	1M NaHPO4	100 ml
	H2O		to 4 L