Recovery of DNA from Low-melting-temperature Agarose Gels: Organic Extraction.

A number of grades of agarose are available in which hydroxyethyl groups have been introduced into the agarose molecule. This substitution causes the agarose to gel at 30 o C and to melt at 65 o C-well below the melting temperature for most DNAs. These properties have been exploited to develop a simple technique for the recovery of DNA from gels (Weislander 1979). 1. Dissolve the low-melting-temperature agarose by heating in electrophoresis buffer to 70 o C. Cool to 37 o C. Add ethidium bromide to a final concentration of 0.5 µg/ml. Pour the gel at 4 o C to ensure that it sets properly. 2. Load the samples of DNA and carry out electrophoresis at 4 o C to ensure that the gel does not melt during the run. The electrophoretic characteristics of low-melting-temperature agarose gels are similar to those of conventional agarose gels. 3. Cut out the desired segment(s) of gel and add about 5 volumes of 20 mM Tris. Cl (pH 8.0) and 1 mM EDTA. 4. Heat for 5 minutes at 65 o C to melt the gel. 5. At room temperature, extract the melted gel slice with an equal volume of phenol. Recover the aqueous phase by centrifugation at 20 o C and re-extract with phenol/chloroform and then with chloroform. 6. Recover the DNA by ethanol precipitation. Usually, the DNA is now pure enough to serve as a substrate for restriction enzymes, ligases, etc. If necessary, the DNA can be further purified by chromatography on DEAE-Sephacel.