Preparative DNA Fragment Isolation from an Agarose Gel
DNA can be easily isolated and purified after size selection on an agarose gel. The fragment of interest is simply cut out of the gel with a razor blade and purified by a number of different methods.The easiest is to use a method that involves first dissolving the agarose slice in a solution at 50°C, then binding the DNA from the melted agarose to a silica-gel membrane.
Prepare an agarose gel in TAE buffer using the four-well combs. (Preparative agarose gels should be run using 1X TAE electrophoresis and gel buffer as the borate in TBE interferes with some purification resin). Load the DNA. To visualize the DNA after staining, do not expose the DNA to shortwave UV light as this will introduce nicks. Visualize the bands with a hand-held long wave UV light and cut out the band with a clean razor blade (Note: place gel on a glass slide to avoid cutting the surface of the transilluminator).
After cutting out the band, follow the procedure for DNA fragment purification using Qiagen QIAquick or Qiaex II purification systems following the manufacturer's procedure (http://www.qiagen.com). Estimate the approximate concentration of the DNA obtained by running 10% of the eluate on an agarose gel against a DNA mass ladder.
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