Molecular Techniques and Methods

Agarose Gel Electrophoresis

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

Agarose is a high molecular weight polysaccharide extracted from the walls of marine red algae. Agarose is an alternating copolymer of 1,3-linked B-D-galactose and 1,4-linked 3,6-anhydro-B-L-galactose. The galactose residues are occasionally substituted with negatively charged groups such as sulfate and pyruvate, giving the agarose fibers a fixed negative charge. Agarose is insoluble in cold water but dissolves readily in boiling water. Upon cooling, the agarose chains form side-by-side aggregates which condense into a three-dimensional, interlocking network held together by noncovalent hydrogen bonds.

Two major types of agarose are available commercially: Unmodified agaroses and Hydroxyethylated agaroses.
Hydroxyethylated agaroses form gels with lower gel strength and significantly lower melting and gelation temperatures than gels cast from unmodified agarose. The hydroxyethylated (low melting point) agaroses are useful for isolating and purifying specific DNA fragments.




MATERIALS AND SOLUTIONS

50 x TAE Buffer (100 ml)
0.4 M Trizma base ------------------------------- 24.22 g
10 mM EDTA-Na2 ------------------------------ 1.86 g
Glacial acetic acid -------------------------------- 6.5 ml
Add distilled H2O to make a final volume of ----- 100 ml
  • Adjust pH to 8.0.


    5 x TBE Buffer (1 L)
    0.32 M Boric acid --------------------------------- 10 g
    0.5 M Trizma base -------------------------------- 30.25 g
    10 mM EDTA-Na2 ------------------------------- 1.86 g
    Add distilled H2O to make a final volume of ----- 1 liter
  • Adjust pH to 8.6.


    Ethidium Bromide Solution (10 mg/ml)
    Ethidium bromide --------------------------------- 100 mg
    Add distilled H2O to make a final volume of ----- 10 ml
  • Store in a brown bottle at 4oC.


    10 X Glycerol Dye Mix (10 ml)
    Glycerol ------------------------------------------ 6 ml
    0.2% Bromophenol blue -------------------------- 0.4 ml of 5% Bromophenol blue
    0.2% Xylene cyanol FF --------------------------- 0.4 ml of 5% Xylene cyanol FF
    10 X TAE (or 10 X TBE) ------------------------ 1 ml
    Distilled H2O ------------------------------------- 2.2 ml


    10 X Ficoll Dye Mix (5 ml)
    50% Ficoll type 400L (MW 400,000) ----------- 2.5 g
    0.14% Bromophenol blue ------------------------ 0.14 ml of 5% Bromophenol blue
    0.14% Xylene cyanol FF ------------------------- 0.14 ml of 5% Xylene cyanol FF
    Add distilled H2O to make a final volume of ----- 5 ml
  • Adjust pH to 8.0 by 5-10 ul of 1 N NaOH.




    PROCEDURES

    1. Weigh out 1 g agarose powder and add to the 100 ml of 1 X TAE (or 1 X TBE) buffer solution in a 500 ml Erlenmeyer flask.

    2. Heat in a microwave oven for 3 minutes.

    3. Swirl the solution gently to release trapped air and resuspend any agarose particles caught on the side of the flask.

    4. Heat in a microwave oven for additional 1 minutes.

    5. Cool down the agarose solution to 50-55oC.

    6. Add 25 ul of Ethidium Bromide Solution (10 mg/ml), and swirl the flask gently to disperse the dye.

    7. Place the comb(s) in the gel box.

    8. Pour the warm agarose solution into the gel box.

    9. After the agarose has set for 30 minutes, place the gel box in the buffer tank.
  • Fill the buffer tanks with fresh running buffer (1 X TAE or 1 X TBE buffer) including ethidium bromide (2.5 ug/ml, or 25 ul/ 100 ml running buffer).

    10. Gently remove the comb(s) by wiggling back and forth and then lifting up.
  • If the gel is not to be used immediately, cover with a piece of plastic wrap.

    11. Prepare DNA sample solution into microfuge tube as follow.

    DNA
    50-500 ng
    TE Buffer
    9 ul
    10 X Glycerol Dye Mix (or, 10 X Ficoll Dye Mix)
    1 ul



    12. Load 10-15 ul DNA sample solution under the buffer in the wells.
  • Place a piece of dark paper beneath the gel form to make the wells more visible.

    13. Turn on the power supply voltage.
  • Apply a voltage of 100-250 volts or 2.5 volts/cm.

    14. Run the electrophoresis for 1-24 hours depending on the type of separation.

    15. If ethidium bromide was not included in the gel matrix, the gel should be stained with ethidium bromide.

    16. Stain the gel in 100 ml of 1 X TAE (or 1 X TBE) and 25 ul of Ethidium Bromide Solution (10 mg/ml) for 15-30 minutes.

    17. Decant the ethidium bromide solution.

    18. Destain the gel in 100 ml 1 X TAE (or 1 X TBE) for 15-30 minutes.

    19. Illuminate analytical gels with short wavelength UV light (254 nm) generated by Mineral-light model R-52.

    20. Illuminate preparative gels with long wavelength UV light (365 nm) generated by Mineral-light model R-56 to prevent nicking of the DNA/dye complexes.

    21. Photograph the gel.




    NOTES

  • Glycerol dye mix may cause edge-tailing of the DNA bands, i.e., the individual bands are U-shaped and trail backwards at each edge.

  • In agarose gels, bromophenol blue coelectrophreses with 200-400 bp DNA; xylene cyanol FF coelectrophreses with 2-4 kb DNA.





    • KIT INFORMATION




    REFERENCES

  • Tietz, D., 1998, Nucleic Acid Electrophoresis, Springer Lab Manual.


  • Please send your comment on this protocol to "editor@MolecularInfo.com".
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