Electrophoresis - Agarose Gels for Duplex DNA
1. Prepare 20X TBE as:
216 g Tris Base
110 g Boric Acid
80 mL 500 mM EDTA, pH 8.0
700 mL ddH2O
Bring volume to 1 L.
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
This page has been viewed 250 times.
eckofoid at ucdavis.edu