Electrophoresis - Agarose Gels for Duplex DNA


Jeff Lawrence

1. Prepare 20X TBE as:
	216 g	Tris Base
	110 g	Boric Acid
	80 mL	500 mM EDTA, pH 8.0
	700 mL	ddH2O
	Mix.  
	Bring volume to 1 L.  
	Autoclave.
2. Mix the following:
	0.40 g 	Agarose
	1.25 mL 	20X TBE (1/2X TBE final)
	48.75 mL 	Water
	1.00 µL	10 mg/mL Ethidium Bromide
3. Melt agarose in the microwave

4. Seal horizontal gel apparatus. Pour molten agarose onto gel plate to a depth of 4 - 8 mm.

5. Insert a comb until its base is 1 mm from the base of the gel. Allow to cool

6. Remove comb and submerge in 0.5X TBE buffer.

7. Prepare 6X Loading Dye as:
	3 mL	100% Glycerol
	3 mL	0.5 M EDTA, pH 8.0
	3 mg	Bromophenol Blue
	3 mg	Xylene Cyanol
	4 mL	Sterile Water
	10 mL	Total Volume
8. Add 1/5 volume 6X Loading Dye to sample. Mix well and pellet in the microfuge.

9. Add sample to well with a yellow tip. Do not overflow well. For large samples, either reduce sample in volume prior to adding dye, or load into multiple wells.

10. Connect electrodes so that the DNA runs to the red (PLUS) electrode.

11. Electrophorese until Bromophenol Blue is near the end of the gel. This dye runs at ~500 bp.

12. Remove gel and visualize bands under uv light.

Last Update: Thursday, 19-Jun-2014 11:50:28 PDT
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Eric Kofoid eckofoid at ucdavis.edu