DNA/RNA Purification from Agarose Gels

As with PAGE electrophoresis, the DNA or RNA resolved into a band on an agarose gel is of high sequence purity, and it is often advantageous to recover this material. As later steps in processing may be sensitive to agarose, its contaminants or the components of the running buffer, it may be necessary to separate the nucleic acid from the matrix material. A variety of techniques have been developed to carry out such separations. In cases where agarose and buffer components will not interfere, low melting agarose gels are used for the electrophoretic separation. The gel is stained and the band(s) of interest excised. The excised material can be melted at 60-65°C, well below the melting temperature of any DNA longer than 30 bases. Processing is then carried out in the melted gel. (See In Gel Enzyme Reactions.)

Use of low melting agarose also facilitates purification from the agarose matrix. The challenge of recovering DNA from a matrix arises from the fact that the nucleic acid molecules are caged in a 3 dimensional network of matrix molecules. Removing the DNA from this cage requires forcing the DNA through the matrix pores. Use of low melting agarose circumvents this problem by disrupting the matrix to release the DNA into the solution, considerably simplifying the physical separation of the two components.

Purification of DNA from Low Melt Agarose Gels

  1. Cast and run a gel, stain and excise band(s).
  1. Add 3 volumes TE buffer to the gel slice (for gels over 2%, use 5 volumes TE).
  1. Melt agarose at 65° for 15-30 minutes.
  1. Add to the melted agarose solution an equal volume of phenol, buffered to pH 8.0 with 0.1M Tris HCl and mix by vortex or vigorous shaking for 10 minutes - the longer it is mixed, the cleaner the final product.
  1. Centrifuge in a microcentrifuge for 15 minutes at 10,000 rpm.
  1. Collect upper aqueous phase. Do not recover any of the white interface material. (More DNA can be recovered by re-extracting the phenol/interface with an equal volume of TE buffer, but this will also carry some agarose into the aqueous phase.)
  1. Precipitate the aqueous fraction(s) with 0.1 volume of 3M sodium acetate and 3 volumes of cold ethanol.
  1. Recover DNA by centrifugation, wash once with cold 70% ethanol and allow to air dry.

DNA purified from low melt agarose by the preceding protocol is sufficiently clean for many purposes. When further purification proves necessary, glass powder elution is an effective method. In this technique, the DNA is bound to finely powdered glass or microscopic glass beads in a high salt suspension. Agarose and other contaminants do not bind to the glass and can be washed away. The DNA is then eluted in water or a low salt buffer. A selection of glass powder elution kits are commercially available. DNA may also be purified from standard agarose gels by this technique, using sodium iodide (NaI) to disrupt the gel matrix.

Glass Powder Elution

While glass powder elution works well for DNA from 200-3000 bp, use of the technique beyond this size range is not advised. Shorter pieces of DNA tend to give poor recoveries. Larger pieces are subject to shearing.

  1. Add 3 volumes of 6M NaI 150mM Na2S03 to the gel slice.
  1. Incubate at 50°C for 5-10 minutes to dissolve the gel, mixing every 2-3 minutes.
  1. Add 5µl of glass powder suspension (1vol glass powder or glass beads + 1vol water)
  1. Mix for 5 minutes to allow DNA to bind.
  1. Pellet glass powder 10 seconds in a microcentrifuge.
  1. Wash the glass powder by suspending in:

50% ethanol
10mM Tris HCl pH 7.5
0.5mM EDTA
50mM NaCl
  1. Pellet washed glass powder.
  1. Elute DNA with 10µl of water or TE buffer.
  1. Incubate 5 minutes at 37°C.
  1. Pellet glass powder and recover DNA in supernatant.

Electroelution

The most popular alternative to glass powder elution for the complete purification of DNA from agarose is electroelution. Because agarose gels are run in a horizontal apparatus, the gel can be manipulated during a pause in the run. This allows variations of electroelution to be performed that are not possible with vertical gels, which are encased in glass plates throughout the entire run.

In the most straightforward form of electroelution, the band is excised from the gel and placed in a bag of dialysis membrane. This bag is then filled with electrophoresis buffer and placed in an electric field. The DNA migrates out of the gel slice and into the buffer, but it is too large to migrate out of the bag. Recovery is then just a matter of collecting the buffer from the bag.

An alternative involves cutting a "trench" into the gel just ahead of the band of interest, and then continuing the electrophoresis until the band is eluted into the trench. Although such technique allows recovery of the band in a small volume of running buffer, it requires exact timing or running the gel on a UV transilluminator, to avoid running the band past the trench.

Alternatively, instead of a trench, a slit can be cut in the gel just ahead of the band, and a piece of DEAE ion exchange paper can be inserted into the gel, so that the band is run onto the paper. The DNA binds tightly to the paper, and there is no need for exact timing and absolutely continuous monitoring of the run. Once all of the band is bound to the paper, recovery is accomplished by washing the paper in a high salt buffer. With this protocol it is often necessary to ethanol precipitate the DNA to remove the elution buffer.

Electroelution into a Dialysis Bag

  1. Cut the band of interest out of the gel, and trim away excess agarose.
  1. Tie two knots in the end of a 1cm diameter dialysis tube, 5cm long. Alternatively, the bag can be closed with a plastic clip.
  1. Place the band in the dialysis tube, add 0.5-1ml of TAE buffer, and seal the bag with knots or a second clip.
  1. Place the bag in a horizontal electrophoresis apparatus, and add 1X TAE until the bag is barely submerged.
  1. Apply a voltage of 2-3 V per centimeter of distance between the electrodes.
  1. Elute for 1 hr per kilobase of target length. For fragments over 5kb, elute overnight.
  1. At the end of the run, reverse the polarity and run at 5v/cm for 1 minute to release any DNA adhered to the inside of the tubing.
  1. Open the bag and recover the TAE. Rinse the bag with 0.5-1ml TAE and pool the TAE fractions.
  1. Precipitate the DNA with 0.1 volume 3M Sodium Acetate and 3 volumes Ethanol.

Electroelution into a Trough

  1. Run the gel until the band of interest is adequately resolved. This is best done in an apparatus which can be mounted on a UV light box, running the gel in buffer containing Ethidium Bromide, and checking the band progress periodically. As an alternative, judge the run by the migration of the tracking dyes, staining the gel in Ethidium Bromide prior to elution to locate band of interest.
  1. Cut a trough 2mm wide just ahead of the band, 2mm wider than the band. Return the gel to the apparatus, and add or remove buffer until the top surface of the gel is barely above the level of the buffer. Fill the trough with buffer and run the gel until the band has entered the trough. If continuous monitoring is not feasible, stop the gel periodically to remove the buffer and refill the trough.

An alternative, more reliable procedure is to cut a slit ahead of the band, and insert a piece of Whatman 3mm paper backed by a piece of dialysis membrane. Upon resumption of electrophoresis, the DNA is trapped against the dialysis membrane and can be easily recovered by eluting or centrifuging the buffer from the Whatman paper.

Electroelution onto DEAE Paper

Anion exchange paper will bind DNA tightly in the relatively low salt environment of an electrophoresis buffer. A strip of DEAE paper, placed in front of a DNA band, will effectively trap all of the DNA in the band. The DNA can be eluted in high purity with high salt.

  1. Run an agarose gel and stain with Ethidium Bromide.
  1. Locate the band of interest and cut slits in the gel just before and just after the band.
  1. Insert a piece of DEAE filter paper into each slit, and return the gel to the electrophoresis chamber.
  1. Continue to run the gel for 10-20 minutes, until the entire band is bound to the paper. The paper inserted above the band prevents any contamination from larger DNA fragments.
  1. Recover the paper, and rinse briefly in electrophoresis buffer. Elute the DNA by placing the paper into 500 µl of 1M NaCl, and heating to 65°C for 30 minutes per kilobase of DNA.
  1. Ethanol precipitate with 1ml of Ethanol and wash pellet twice with 70% Ethanol.

NEXT TOPIC: In Gel Enzyme Reactions

/ Products Related to this Discussion: /
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EDTA - ULTRA PURE
Chelating agent added to electrophoresis buffers.