R E C I P E S

III. Agarose Gel Electrophoresis

2.0% Agarose

Makes 200 ml. Use fresh or store jelled at room temperature (several weeks).

  1. Add 4.0 g agarose (electrophoresis grade) to 200 ml 1X TBE electrophoresis buffer in a 600 ml beaker or Erlenmeyer flask.
  2. Stir to suspend agarose.
  3. Cover beaker with aluminum foil, and heat in boiling-water bath (double boiler) or on hot plate until all agarose is dissolved (approximately 10 minutes).
    or
    Heat uncovered in a microwave oven at high setting until all agarose is dissolved (3-5 minutes per beaker).
  4. Swirl solution and check bottom of beaker to insure that all agarose has dissolved. (Just prior to complete dissolution, particles of agarose appear as translucent grains.) Reheat for several minutes if necessary.
  5. Cover with aluminum foil, and hold in a hot-water bath (at about 60C) until ready for use. Remove any "skin" of solidified agarose from surface prior to pouring.

Notes:

i. Samples of agarose powder can be preweighed and stored in capped test tubes until ready for use.

ii. Solidified agarose can be stored at room temperature and then remelted over a boiling-water bath (15-20 minutes) or in a microwave oven (3-5 minutes per beaker) prior to use. Always loosen cap when remelting agarose in a bottle.

iii. When remelting agarose evaporation will cause the concentration to increase. If necessary, compensate by adding back a small volume of water.


1 g/ml Ethidium Bromide Staining Solution

Makes 500 ml. Store in dark at room temperature (indefinitely).

CAUTION: Ethidium bromide is a mutagen by the Ames microsome assay and a suspected carcinogen. Wear rubber gloves when preparing and using ethidium bromide solutions.

  1. Add 100 l of 5 mg/ml ethidium bromide to 500 ml deionized or distilled water.
  2. Store in unbreakable bottles (preferably opaque). Label bottles "CAUTION: Ethidium Bromide. Mutagen and cancer-suspect agent. Wear rubber gloves when handling."

Note: Ethidium bromide is light sensitive; store in dark container or wrap container in aluminum foil.


Loading Dye

Makes 100 ml. Store at room temperature (indefinitely).

0.25 g bromophenol blue (m.w. 669.96)
0.25 g xylene cyanol (m.w. 538.60)
50.00 g sucrose (m.w. 342.30) (or 50 ml of glycerol)
1.00 ml 1M Tris (pH 8.0)

If using sucrose:

  1. Dissolve bromophenol blue, xylene cyanol, sucrose and Tris in 60 ml deionized or distilled water.
  2. Add deionized or distilled water to make 100 ml total solution.

If using glycerol:

  1. Dissolve xylene cyanol, bromophenol blue, and Tris in 49 ml of deionized or distilled water.
  2. Stir in 50 ml of glycerol to make 100 ml total solution.


0.2% Methylene Blue Stock Solution

Makes 100 ml. Store at room temperature (indefinitely).

  1. Weigh out 0.2 g methylene blue-trihydrate (m.w. 373.9) and add to 100 ml of distilled water.
  2. Stir until completely dissolved.

0.025% Methylene Blue Staining Solution

Makes 500 ml. Store at room temperature (indefinitely).

  1. Add 62.5 ml of 0.2% methelyene blue stock solution to 437.5 ml of distilled water.
  2. Stir to mix.


pBR322/
BstN I Size Markers (0.1 g/l)

Either buy pBR322 pre-cut with restriction enzyme BstN I from New England Biolabs (#N3031) or prepare it according to the following protocol:

Makes 1 ml
Store at -20 C (1year).

  1. Add 10 l of a solution of 10 g/l pBR322 to 75 l distilled, autoclaved water.
  2. Add 10 l 10x buffer (provided by supplier of the enzyme).
  3. Add 5 l BstN I restriction enzyme, and incubate at 60C for 60 min.
  4. Electrophorese 5 l (plus 1 l loading dye) in a 1-2% agarose gel to check for complete digestion. Exactly 5 bands should be visible, corresponding to 1,857 bp, 1,058 bp, 929 bp, 383, and 121 bp. Any additional bands indicate incomplete digestion; add additional enzyme and incubate again at 60C.


10X Tris/Borate/EDTA (TBE) Electrophoresis Buffer

Makes 1 liter. Store at room temperature (indefinitely).

  1. Weigh out:
    1 g NaOH (m.w. 40.00)
    108 g Tris base (m.w. 121.10)
    55 g boric acid (m.w. 61.83)
    7.4 g ethylene diamine tetraacetic acid (EDTA, disodium salt, m.w. 372.24)
  2. Add all dry ingredients to 700 ml deionized or distilled water in a 2 l flask.
  3. Stir to dissolve, preferably using a magnetic stir bar.
  4. Add deionized water to bring total solution to 1 liter.


1X TBE Electrophoresis Buffer

Makes 10 liters. Store at room temperature (indefinitely).

  1. Into a spigoted carboy, add 9 liters deionized or distilled water to 1 liter of 10X TBE electrophoresis buffer.
  2. Stir to mix.



2000, DNA Learning Center, Cold Spring Harbor Laboratory
Noncommercial, educational use only.
Kits available from Carolina Biological Supply Company.