Hello, I'm Meghan, I'm a research scientist at Addgene, and today I'm going to show you how to excise
a DNA band from an agarose gel. Now this video begins after a DNA has already been
prepared and separated on an agarose gel. So if you need a refresher please refer to our
agarose gel electrophoresis protocol video. For this experiment, we will need our agarose gel, a
gel imager, a UV resistant face shield, a sterile razor blade and tweezers, micro centrifuge tubes, a
heat block, vortex, centrifuge, and a commercially available gel extraction kit. Since we know our
plasmid sequence we can pick enzymes that cut our DNA at specific locations to generate fragments of
a known size, we can then run these fragments on an agarose gel alongside a controlled DNA ladder
of known sizes, and determine which fragment we want based on where it runs in the gel. By
extracting only the band of the desired size from the gel, we are able to isolate our desired product from
the rest of the plasmid backbone. After extraction, the DNA is purified and can then be used
For an array of downstream applications, such as:
molecular cloning or next-generation sequencing.
Before we can remove our DNA band from the gel, we first need to be able to visualize the bands.
To start, remove the gel from the plastic tray you used during electrophoresis, as the plastic
will block the UV light from passing into the gel and lighting up your DNA. Next, carefully place the
gel into the gel imager. Before working with UV light, always wear a UV light blocking protective
face shield and cover exposed skin. Different UV imagers will have different instructions
for band excision
The gel we use contains ethidium bromide, a stain that binds to DNA and causes it to fluoresce under the UV
light emitted by the gel imager. Pull the tray out slightly to prepare to cut the bands. If the
bands are not easily visible when the tray is out, try to block out surrounding light by closing the
shades or turning off the lights. Once you have located your band of choice, use a sterile razor
blade to cut around the band down through the gel. Try to cut out as close to the band as possible to
avoid contamination from other bands, and limit the excess gel that will need to be processed
later. Do not cut with too much pressure as you do not want to scratch the gel imager. When you
have successfully cut around the band, separate the extracted piece from the rest of the gel
and place into a micro centrifuge tube. A set of sterile tweezers or stall pipette tip can be used
to carefully maneuver the gel piece into the tube. Next, the gel fragment must be weighed in order to
determine the amount of each buffer to add during the DNA isolation step. First, blank the scale with
an empty micro centrifuge tube. Once the scale is blanked, weigh the micro centrifuge tube containing
the gel slice. Now, we are ready to purify the DNA from the remaining agarose gel matrix. For this
process we use commercially available purification kits. These kits work by first breaking down the
gel with heat and mild vortexing to release the DNA fragments,
binding the DNA to a centrifugal filter,
purifying the DNA through several wash steps, and finally eluding the DNA from the column.
Once the DNA has been isolated from the gel,
you should run a small a aliquot on a new agarose gel.
Thank you for watching our video on how to purify DNA
from an agarose gel. We hope you find this protocol useful as you perform the procedure yourself. For
more protocol videos visit the protocols playlist on Addgene's YouTube channel. To browse written
protocol content visit Addgene.org\protocols
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