Biotechnology Index Glossary
Isolation of DNA from Agarose and Polyacrylamide Gels
In addition to its importance as an analytical tool, gel electrophoresis is widely used for isolating and then purifying specific fragments of DNA, usually in preparation for subcloning. Hence, this is a very commonly required procedure.
Several techniques can be used to purify DNA from agarose gels, and choosing between them is, to some extent, a matter of personal preference. They all start out by excising the desired "band" from an ethidium-stained gel viewed with a UV transilluminator. Because UV light can fragment DNA, it is best to work expeditiously and keep exposure time to a minumum. Cut out the desired piece of agarose using a razor blade or scalpel blade, and try to get as little extra agarose as possible. The block of agarose containing DNA is then subjected to any of the following.
Electroelution: The block of agarose is placed in a piece of dialysis tubing with a small amount of fresh electrophoresis buffer, the ends sealed with clamps, and the bag placed into an electrophoresis chamber. Application of current will cause the DNA to migrate out of the agarose, but it will be trapped within the bag. Progress can be monitored using a transilluminator, as shown below. When the DNA is out of the agarose, the flow of current is reversed for a few seconds to knock the DNA off of the side of the tubing. The buffer containing the DNA is then collected and the DNA precipitated with ethanol.
Electroelution is more time consuming than some of the other techniques, but works well and is probably the best technique for recovery of large (> 5 kb) fragments of DNA.
Binding and elution from glass or silica particles: In an environment of high salt and neutral or low pH, DNA binds avidly to glass, silica gel or diatomaceous earth. This phenomenon can be exploited to purify DNA from solutions containing impurities such as agarose. Typically, a slice of agarose containing the DNA of interest is "melted" by incubation in a solution containing chaotropic salt (e.g. sodium iodide) at a pH of 7.5 or less. Glass powder or silica gel is then added and the suspension is mixed to allow adsorption of DNA. The particles can then be recovered from the original liquid and washed by centrifugation and resuspension in high-salt-ethanol buffer. Finally, the pellet is resuspended in a solution with low or no salt at basic pH, the free particles pelleted by another centrifugation, and the DNA-containing supernate recovered.
The glass or silica particles used for this technique can be prepared in house or, more conveniently, purchased from a number of suppliers.
Electrophoresis onto DEAE-cellulose membranes: At low concentrations of salt, DNA binds avidly to DEAE-cellulose membranes. Fragments of DNA are electrophoresed in a standard agarose gel until they resolve adequately. One then makes a slit in the gel slightly ahead of the fragment(s) of interest and resumes electrophoresis until all of that fragment has migrated and stuck onto the membrane. The membrane is then removed, washed free of agarose in low salt buffer (150 mM NaCl, 50 mM Tris, 10 mM EDTA), then incubated for about 30 minutes at 65 C in high salt buffer (1 M NaCl, 50 mM Tris, 10 mM EDTA) to elute the DNA. Progress in binding DNA to the membrane and eluting it can be monitored with UV light to detect the ethidium bromide bound to DNA. After elution, DNA is precipitated with ethanol. This procedure is simple and provides very clean DNA. However, fragments larger than about 5 kb do not elute well from the membrane.
Low melting point agarose: Agarose can be purchased that melts at about 65 C, which is well below the melting temperature of all but very small fragments of double-stranded DNA. After electrophoresing in such low melting temperature agarose, the appropriate fragment is excised, and the agarose block is added to a small quantity of buffer and incubated at 65 C to melt the agarose. An alternative technique is to digest the agarose with agarase. One can then extract the melted or digested agarose from the mixture with phenol and precipitate the DNA with ethanol.
A commonly-used means of recovering DNA from polyacrylamide gels is by the so-called "crush and soak" method. The slice of polyacrylamide containing DNA is crushed in a microcentrifuge using a plastic pipet tip, and incubated with constant shaking in elution buffer (high salt) at 37C for several hours. The polyacrylamide pieces are then eliminated by centrifugation or by passing the mixture through a plug of siliconized glass wool. Finally, DNA is recovered by ethanol precipitation.
DNA can also be recovered from polyacrylamide by use of certain types of silica gel particles, as described above for recovery from agarose. However, small (< 100 bp) fragments of DNA are very difficult to elute from standard glass particles.
Back to the index of Gel Electrophoresis of DNA and RNA
Last updated on January 9, 2000
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