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Electrophoresis - Agarose Gels for Single Stranded DNA

Electrophoresis - Agarose Gels for Single Stranded DNA

Jeff Lawrence

1. Prepare 50X TAE as:
 	242 g	Tris Base 	57.1 mL	Glacial Acetic Acid 	100 mL	500 mM EDTA, pH 8.0 	600 mL	ddH2O 	Mix.   	Bring volume to 1 L.   	Autoclave. 
2. Mix the following:
	0.40 g		Agarose 	4.00 mL 	50X TBE (4X TAE final) 	46.00 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 4X TAE 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. Electrophorese until Bromophenol Blue is near the end of the gel. This dye runs at ~800 b.

11. Remove gel and visualize bands under uv light.