DNA Transfer - Dot Blots

Jeff Lawrence

1. Wet a Nitrocellulose filter in water, then 1 M NH4OAc.

2. Centrifuge 400 µL cells. Resuspend cells in 200 µL of:

	50 mM 	Tris, pH 8.0	5 mL	1 M Tris, pH 8.0
	50 mM 	EDTA	10 mL	500 mM EDTA, pH 8.0
			85 mL	ddH₂O

3. Add 15 µL 5 mg/mL Proteinase K. Incubate at 37° for 15 min.

4. Add 25 µL 10% SDS. Incubate at 37° for 15 min. Chill samples on ice.

5. Add 22 µL 5 M NaOH. Incubate at 22° for 15 min. Mix by vortexing. Chill on ice.

6. Add 140 µL 3 M NH4OAc.

7. Load filter onto DotBlot manifold and prewet wells with 100 µL 1M NH4OAc. Add samples to wells.

8. Draw samples through he wells and onto the membrane with vacuum for 30 min.

9. Remove filter and float on 6X SSC for 10 min. Air dry. 20XSSC is:

	3.0 M	NaCl
	0.3 M	NaCitrate

10. Bake at 80° for 90 min.

Last Update: Thursday June 19 2014

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Eric Kofoid eckofoid at ucdavis.edu