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Agarose Gel Electrophoresis for the Separation of DNA Fragments

互联网2013-11-13

1403

实验材料

Name	Company	Catalog Number	Comments
Agarose I	Amresco	0710
Boric acid	Sigma	B7901
Bromophenol blue	Sigma	B8026
EDTA	Sigma	E9884
Ethidium bromide	Sigma	E7637	Carcinogenic—needs to be disposed of as hazardous waste
Glacial acetic acid	Fisher	BP2401-212	Corrosive
Glycerol	Fisher	G33-1
Xylene cyanol FF	Sigma	X4126
Tris base	Sigma	T1503
Investigator/FX gel documentation system	Fotodyne
Owl Easycast B1 mini gel electrophoresis system	Thermo Scientific	B1-PTM
EPS 301 power supply	GE Healthcare	18-1130-01

实验步骤

1. Preparation of the Gel

1) Weigh out the appropriate mass of agarose into an Erlenmeyer flask. Agarose gels are prepared using a w/v percentage solution. The concentration of agarose in a gel will depend on the sizes of the DNA fragments to be separated, with most gels ranging between 0.5%-2%. The volume of the buffer should not be greater than 1/3 of the capacity of the flask.

2) Add running buffer to the agarose-containing flask. Swirl to mix. The most common gel running buffers are TAE (40 mM Tris-acetate, 1 mM EDTA) and TBE (45 mM Tris-borate, 1 mM EDTA).

3) Melt the agarose/buffer mixture. This is most commonly done by heating in a microwave, but can also be done over a Bunsen flame. At 30 s intervals, remove the flask and swirl the contents to mix well. Repeat until the agarose has completely dissolved.

4) Add ethidium bromide (EtBr) to a concentration of 0.5 μg/ml. Alternatively, the gel may also be stained after electrophoresis in running buffer containing 0.5 μg/ml EtBr for 15-30 min, followed by destaining in running buffer for an equal length of time.

Note: EtBr is a suspected carcinogen and must be properly disposed of per institution regulations. Gloves should always be worn when handling gels containing EtBr. Alternative dyes for the staining of DNA are available; however EtBr remains the most popular one due to its sensitivity and cost.

5) Allow the agarose to cool either on the benchtop or by incubation in a 65 °C water bath. Failure to do so will warp the gel tray.

6) Place the gel tray into the casting apparatus. Alternatively, one may also tape the open edges of a gel tray to create a mold. Place an appropriate comb into the gel mold to create the wells.

7) Pour the molten agarose into the gel mold. Allow the agarose to set at room temperature. Remove the comb and place the gel in the gel box. Alternatively, the gel can also be wrapped in plastic wrap and stored at 4 °C until use (Fig. 1).

2. Setting up of Gel Apparatus and Separation of DNA Fragments

1) Add loading dye to the DNA samples to be separated (Fig. 2). Gel loading dye is typically made at 6X concentration (0.25% bromphenol blue, 0.25% xylene cyanol, 30% glycerol). Loading dye helps to track how far your DNA sample has traveled, and also allows the sample to sink into the gel.

2) Program the power supply to desired voltage (1-5V/cm between electrodes).

3) Add enough running buffer to cover the surface of the gel. It is important to use the same running buffer as the one used to prepare the gel.

4) Attach the leads of the gel box to the power supply. Turn on the power supply and verify that both gel box and power supply are working.

5) Remove the lid. Slowly and carefully load the DNA sample(s) into the gel (Fig. 3). An appropriate DNA size marker should always be loaded along with experimental samples.

6) Replace the lid to the gel box. The cathode (black leads) should be closer the wells than the anode (red leads). Double check that the electrodes are plugged into the correct slots in the power supply.

7) Turn on the power. Run the gel until the dye has migrated to an appropriate distance.

3. Observing Separated DNA fragments

1) When electrophoresis has completed, turn off the power supply and remove the lid of the gel box.

2) Remove gel from the gel box. Drain off excess buffer from the surface of the gel. Place the gel tray on paper towels to absorb any extra running buffer.

3) Remove the gel from the gel tray and expose the gel to uv light. This is most commonly done using a gel documentation system (Fig. 4). DNA bands should show up as orange fluorescent bands. Take a picture of the gel (Fig. 5).

4) Properly dispose of the gel and running buffer per institution regulations.

4. Representative Results

Figure 5 represents a typical result after agarose gel electrophoresis of PCR products. After separation, the resulting DNA fragments are visible as clearly defined bands. The DNA standard or ladder should be separated to a degree that allows for the useful determination of the sizes of sample bands. In the example shown, DNA fragments of 765 bp, 880 bp and 1022 bp are separated on a 1.5% agarose gel along with a 2-log DNA ladder.

1. Sambrook, J. & Russell, D.W. Molecular Cloning, 3^rd edition (2001).

2. Kirkpatrick, F.H. Overview of agarose gel properties. Electrophoresis of large DNA molecules: theory and applications 9-22 (1991).

3. Helling, R.B., Goodman, H.M., & Boyer, H.W. Analysis of endonuclease R•Eco RI fragments of DNA from lambdoid bacteriophages and other viruses by agarose-gel electrophoresis. J. Virol. 14, 1235-1244 (1974).

4. Smith, S.B., Aldridge, P.K., & Callis, J.B. Observation of individual DNA molecules undergoing gel electrophoresis. Science. 243, 203-206 (1989).

5. Aaji, C. & Borst, P. The gel electrophoresis of DNA. Biochim. Biophys. Acta. 269, 192-200 (1972).

6. Lai, E. Birren, B.W. Clark, S.M., Simon, M.I., & Hood L. Pulsed field gel electrophoresis. Biotechniques. 7, 34-42 (1989).

7. Devor, E.J. IDT tutorial: gel electrophoresis http://cdn.idtdna.com/Support/Technical/TechnicalBulletinPDF/Gel_Electrophoresis.pdf

8. Serwer, P. Agarose gels: properties and use for electrophoresis. Electrophoresis. 4, 375-382 (1983).

9. Dea, I.C.M., McKinnon, A.A., & Rees, D.A. Tertiary and quaternary structure and aqueous polysaccharide systems which model cell wall adhesion: reversible changes in conformation and association if agarose, carrageenan and galactomannans. J. Mol. Biol. 68, 153-172 (1972).

10. Sharp, P.A., Sugden, B., & Sambrook, J. Detection of two restriction endonuclease activities in H. parainfluenzae using analytical agarose-ethidium bromide electrophoresis. Biochemistry. 12, 3055-3063 (1973).

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