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 Addgene - a better way to share science
