DNA/RNA Purification from Agarose Gels - 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.
  2. Tie two knots in the end of a 1cm diameter dialysis tube, 5cm long. Alternatively, the bag can be closed with a plastic clip.
  3. Place the band in the dialysis tube, add 0.5-1ml of TAE buffer, and seal the bag with knots or a second clip.
  4. Place the bag in a horizontal electrophoresis apparatus, and add 1X TAE until the bag is barely submerged.
  5. Apply a voltage of 2-3 V per centimeter of distance between the electrodes.
  6. Elute for 1 hr per kilobase of target length. For fragments over 5kb, elute overnight.
  7. 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.
  8. Open the bag and recover the TAE. Rinse the bag with 0.5-1ml TAE and pool the TAE fractions.
  9. 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.
  2. 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.
  2. Locate the band of interest and cut slits in the gel just before and just after the band.
  3. Insert a piece of DEAE filter paper into each slit, and return the gel to the electrophoresis chamber.
  4. 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.
  5. 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.
  6. Precipitate with 1ml of ethanol and wash pellet twice with 70% ethanol.

 

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