Biotech Glossary | Bioinformatics | Lab Protocol | Notes | Malaysia University |
CONTRIBUTOR: The University of Maryland Baltimore County Applied Molecular Biology Program
PROCEDURE:
A. Gel Preparation and Electrophoresis
Ficoll 400
1. Prepare 100 ml of 0.7% Agarose Solution in a glass beaker or flask.
2. Microwave or stir on a hot plate until Agarose is dissolved and solution is clear.
3. OPTIONAL: Before the Agarose Gel solidifies, add Ethidium Bromide to a final concentration of 0.5 μg/ml (if Ethidium Bromide is not added now, follow Section B of this protocol after completing Section A).
4. Allow solution to cool to approximately 50°C before pouring into a gel casting.
5. Prepare gel tray by sealing ends with tape or other custom-made dam.
6. Place comb in gel tray approximately 1 inch from one end of the tray and position the comb vertically such that the teeth are about 1 to 2 mm above the surface of the tray.
7. Pour gel solution into tray to a depth of approximately 5 mm. Allow gel to solidify (approximately 20 min at room temperature).
8. To run the gel, gently remove the comb, place the tray in electrophoresis chamber, and cover (until wells are submerged) with electrophoresis buffer (use the same solution used to prepare the agarose gel. i.e., either 1X TAE or 1X TBE).
9. Excess agarose can be stored at room temperature and re-melted in a microwave.
10. To prepare samples for electrophoresis, combine 1 μl of 6X Gel Loading Dye for every 5 μl of DNA solution and mix well.
11. For mini-gels load 5 to 12 μl of DNA per well.
12. Electrophorese at 50-150 V until the dye markers have migrated an appropriate distance (see Hint #2).
B. Staining an Agarose Gel with Ethidium Bromide
1. Remove the gel from the casting.
2. Completely submerge the gel in a 0.5 μg/ml Ethidium Bromide solution.
3. Incubate the gel in Ethidium Bromide for an appropriate amount of time to properly stain the DNA (see Hint #3).
4. Extensively wash the gel in ddH2O.
SOLUTION:
Agarose Solution
Typically the Agarose content ranges from 0.7% to 1.0% (w/v)
in 1X TBE Buffer or TAE Buffer
0.7% (w/v) Agarose 
Ethidium Bromide
10 mg/ml Ethidium Bromide
TBE Buffer (10X)
890 mM Boric Acid
20 mM EDTA, pH 8.0
890 mM Tris
Gel Loading Buffer (6X)
0.25% (w/v) Bromophenol Blue
15% (v/v) Ficoll 400
120 mM EDTA
0.25% (w/v) Xylene Cyanol FF
TAE buffer (50X)
Working concentrations:
40 mM Tris:Acetate
37.2 g Disodium EDTA
57.1 ml Glacial Acetic Acid
Add ddH2O to 1 liter
242 g Tris base
pH 8.5
2 mM EDTA
REAGENTS AND CHEMICALS:
EDTA
Tris Base
Xylene Cyanol FF
Bromophenol Blue
Ethidium Bromide
Glacial Acetic Acid
Boric Acid
Agarose