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
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Electroelution into a 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.
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