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Agarose Gel Electrophoresis
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| Protocol Information |
| Title |
Agarose Gel Electrophoresis |
| Contributor |
Barry Hudson, Ph.D. |
| Overview |
| The electrophoresis of DNA is an essential technique in molecular biology, relying on the negative charge of DNA for size separation in a sieving matrix. Traditionally, electrophoresis is performed using agarose (an extract of seaweed) and a Tris buffering solution. Here, we present both the Tris Buffered Solution Method (developed 30 years ago) and an alternative method, Salt Buffer for Rapid Electrophoresis (recently developed by Brody and Kern). |
| Procedure |
- For a 1% agarose gel, weigh out 1g of agarose into a flask and add 100ml of 1 x TBE or TAE. There are a variety of agarose products available commercially (they have different melting properties). For separating simple DNA fragments of 0.1–10 kb, normal grade or low melting temperature agarose is adequate. The following table gives a guide for size separation of DNA fragments:
Agarose concentration (%) Separation range of linear DNA
0.5 2 – 40 kb
1 0.4 - 7
1.5 0.2 - 4
2 0.1 - 3
- Heat solution in a microwave or boiling water bath until agarose is completely dissolved.
- Allow to cool in a water bath set at 50 – 55 C for 10 min.
- Prepare gel casting tray by sealing ends of gel chamber with tape or appropriate casting system. Place appropriate number of combs in gel tray.
- Add 5 ul of ethidium bromide to cooled gel and pour into gel tray. Allow to cool for 15-30 min at room temperature. Gels can also be placed in a cold space and used the following day.
- Remove comb(s), place in electrophoresis chamber and cover with buffer (TAE or TBE as used previously).
- Add loading buffer to samples. As a guideline, add 1.5 ul of 10x Loading Buffer to a 20-25 ul PCR/DNA solution. For more concentrated DNA solutions (e.g. plasmids), prepare tubes with 8 ul of 2x Loading Buffer and 2 ul of plasmid.
- Load DNA and standard (Ladder) onto gel.
- Electrophorese at 100V for 1 h.
- Visualize DNA bands using UV lightbox or gel imaging system.
Alternative Protocol
- Prepare gel as above, substituting the following for TAE/TBE
A. 10mM sodium boric acid (Borax, Na2B4O7) for DNA fragments from 100bp-5kb
B. 5mM lithium acetate for DNA fragments larger than 5kb
C. 1mM lithium boric acid (lithium tetraborate) for separating ssDNA and small DNA fragments
- After casting and setting gel, add the same buffer used for the gel as the running buffer.
- Electrophorese at 10-35 V/cm (100-500V)
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| Reagents/Solutions |
- 50 x TAE : 242g Tris base, 57.1 ml of glacial Acetic acid, 100ml of 0.5M EDTA (pH 8.0), Make up to 1 L with water
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5 x TBE: 54g Tris base: 27.5g of boric acid: 20ml of 0.5M EDTA (pH 8.0), Make up to 1 L with water
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20 x Sodium Boric Acid: 40.2g Borax Anhydrous, Make up to 1 L with water
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20x Lithium Acetate: 6.6g lithium acetate, Make up to 1 L with water
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20x Lithium Boric Acid: 3.38g lithium tetraborate, Make up to 1 L with water
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| References |
- Ogden, R.C., and Adams, D.A., (1987) Electrophoresis in agarose and acrylamide gels. Meth. Enzymol. 152, 61-87
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Brody, JR and Kern SE (2004) Sodium boric acid: a Tris-free, cooler conductive medium for DNA electrophoresis. Biotechniques 36(2):214-6
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Brody, JR and Kern SE (2004) History and principles of conductive media for standard DNA electrophoresis. Anal Biochem. 333(1):1-13. Review.
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Brody, JR and Kern SE (2004) Ultra-fast high-resolution agarose electrophoresis of DNA and RNA using low-molarity conductive media. Biotechniques 37(4):598, 600, 602.
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| Tips |
- If you’re worried using ethidium bromide, Invitrogen sells SYBR Safe DNA Stain, a safer alternative.
- If you prefer to try a pre-made version of the above buffers, TBE and TAE are available from many common lab suppliers in bulk. In addition, Sodium Boric Acid, Lithium Acetate and Lithium Boric Acid are available from Faster Better Media.
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