Conventional methods for the agarose gel extraction of DNA

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Electroelution using the dialysis bag
Electroelution using the dialysis bag (D9777). Credit: Colorado State University

Agarose gel extraction can be made easy by using commercially available gel extraction kits or advanced electrophoretic apparatuses that allow the visualization of DNA bands during the electrophoresis. Conventional methods include dialysis of the sliced gel containing DNA by applying an electric current while some other methods include spin columns and Sephadex G10 column chromatography.

Using dialysis bag

Agarose gel extraction using a dialysis bag is an electroelution process. It involves the elution of DNA by applying an electric current. The principle is that the electric field causes the migration of the DNA band out of the gel. The DNA fragment will be trapped in the dialysis membrane. At first, you need to freeze the gel piece at -20°C for 30 minutes to make it easier to handle.

Steps involved

Cut the dialysis bag (Sigma D9777) of 5 cm and activate it by boiling water for 10 minutes. After activation, rinse it inside and out with distilled water and subsequently rinse it with 0.5 × TBE buffer. Seal one end of the dialysis bag with a dialysis clip (Sigma Z370967) and insert the frozen gel inside the tube.

Add 200-400 µl of 0.5 × TBE buffer and seal the other end of the tube with the second dialysis tube. After that, immerse the dialysis bag into the electrophoresis tank containing the electrophoresis buffer. The DNA band should be parallel to the electrodes and apply an electric field of 5 V/cm or 100 V.

You can track the DNA migration using a UV lamp. It will take 10-15 minutes for DNA to migrate out of the gel. The migrated DNA will adhere to the dialysis membrane towards the positive end. It will stay inside but will not migrate out of the dialysis bag.

Gently reverse the direction of the current to knock the DNA off of the dialysis membrane. After that, collect the buffer from the tube and extract the DNA using phenol/chloroform extraction and ethanol precipitation. Dissolve the precipitated DNA in an appropriate volume (10 µl) of TE buffer or water.

The DNA pellet is small in amount and may not be visible. The final purified sample can easily be used in downstream applications. Though it seems an easy process, agarose gel extraction using a dialysis bag is not that easy protocol. It is time-consuming and still involves phenol/chloroform extraction followed by ethanol precipitation.

Using DEAE-Cellulose paper strip

Agarose gel extraction using DEAE-cellulose paper strip is a combination of electroelution as well as a spin column. In this procedure, make a slit immediately below the band of desired DNA fragment using a surgical blade. But don’t remove the band just make a slit below it. After that, cut a small piece of DEAE-cellulose paper of size 3 mm x 10 mm (same size as the slit).

Insert the paper into the slit and return the gel into the electrophoresis tank. Run the electrophoresis for 2-5 minutes. During the electrophoresis, DNA will migrate out of the band and will adhere to the filter paper. DNA will be eluted out of the paper strip using a spin column. The UV lamps can be used during the process to ensure that the DNA has adhered to the paper. Create a tiny hole at the center of the bottom of the 500 µl microfuge tube by using a needle from inside to out.

Electrophoresis on DEAE-cellulose paper strip
Electrophoresis on DEAE-cellulose paper strip. Credit: Colorado State University

Take the paper strip out of the gel and place it (DNA side facing down) into the 500 µl microfuge tube. Place the 500 µl tube into a 1.5 ml Eppendorf tube. Add 100 µl of TE buffer to the 500 µl tube and spin the tube at 5,000-10,000 rpm for 30 seconds. After centrifugation, DNA will be eluted into the collection tube (1.5 ml). Proceed with the phenol/chloroform extraction protocol followed by ethanol precipitation. These later steps concentrate recovered DNA and the DNA sample can be resuspended in a TE buffer of about 10 µl.

Using spin column

Agarose gel extraction can also be done using an improvised spin column. The use of the spin column is an inexpensive and simple method for the isolation of DNA from agarose gel. The fact is that the DNA fragment is trapped in the agarose gel which is a solution. So, by applying a centrifugal force, it is possible to squeeze DNA out of the gel. However, for this protocol, agarose gel concentration should be 0.3-0.7 %. The lower concentration makes it easier for DNA to squeeze out of the gel.

However, you should not be confused with the spin column used in the commercially available gel extraction kits. These kits also use spin columns but those columns are fitted with a built-in silica membrane to trap the DNA that can later be eluted with elution buffer.

Steps involved

Make a hole in the bottom of a 500 µl centrifuge tube using a 22G syringe needle. The hole should be made from inside to outside. After that, put some glass wool to the bottom of the tube about 4 mm in thickness. Absorbent cotton from cotton swabs can also be used alternative to glass wool. Place the piece of the gel containing desired DNA into the 500 µl tube and cap the tube.

Gel extraction using an improvised spin column
Gel extraction using an improvised spin column. Credit: Sun et al., 2012

Place the 500 µl tube into the 1.7 ml Eppendorf tube and placed it into the centrifuge machine. After that, centrifuge at 5,000-10,000 rpm for 5-10 minutes. The length of centrifugation can be varied depending upon the length of DNA fragments; for 1kb or less use 5,000 rpm for 5 minutes, for 1-2 kb DNA use 10,000 rpm for 5 minutes, and for 2-3 kb fragments use 10,000 rpm for 10 minutes.

Centrifugation spins down the DNA from the gel along with aqueous components that will be collected into the 1.7 ml Eppendorf tube. Thus eluted DNA samples can easily be used for downstream processes. Elution volume is normally 15-30 µl depending upon the amount of agarose gel-excised and the recovery rate is 30-60 % of the amount of DNA in the gel piece. The lower DNA fractions can give rise to an 80 % recovery rate.

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