DNA Transfer - Southern Method

Jeff Lawrence

1. Following electrophoresis, place gel in 500 mL of:

	0.25 M	HCL	10 mL	HCL
			490 mL	ddH₂O

2. Rinse at 22° with agitation for 20 min, until bromophenol blue turns yellow.

3. Decant solution and rinse chamber with water. Add 500 mL of:

	0.5 M	NaOH	10 g	NaOH
	1.0 M	NaCl	29 g	NaCl
			500 mL	ddH₂O

4. Decant solution and rinse chamber with water. Add 500 mL of:

	0.5 M	Tris, pH 7.0	30 g	Tris Base
	1.5 M	NaCl		44 g	NaCl
				900 mL	ddH₂O
				Adjust pH to 7.0 with HCL

5. Fill a tray with 500 mL 4X SSC. Place a glass plate over the tray and drape Whatman 3MM paper over two edges to form a wick.

6. Place gel on Whatman paper wick. Roll out air bubbles with a pipet.

7. Soak Nylon and 1 piece of Whatman 3MM in 4X SSC. Place cut Nylon filter on gel. Clip the upper left-hand corner to orient.

8. Cover the gel and Nylon with wet piece of Whatman 3MM paper.

9. Cover all areas of the gel not to be transferred with plastic wrap.

10. Place a stack of paper towels on to of gel stack. Weigh down slightly.

11. Transfer DNA overnight. Remove filter and air dry.

12. Crosslink DNA by exposure to uv light for 30 sec.

Last Update: Thursday, 12-Dec-2024 00:55:26 UTC

This page has been viewed [an error occurred while processing this directive] times.

Eric Kofoid eckofoid at ucdavis.edu