Pellet pestle homogenization of agarose gel slices at 45 degrees C for deoxyribonucleic acid extraction

A simple method for extracting DNA from agarose gel slices is described. Th e extraction is rapid and does not involve harsh chemicals or sophisticated equipment. The method involves homogenization of the excised gel slice (in Tris-EDTA buffer), containing the DNA fragment of interest, at 45 degreesC in a microcentrifuge tube with a Kontes pellet pestle for I min. The "homo genate" is then centrifuged for 30 s and the supernatant is saved. The "hom ogenized" agarose is extracted one more time and the supernatant obtained i s combined with the previous supernatant. The DNA extracted using this meth od lent itself to restriction enzyme analysis, ligation, transformation, an d expression of functional protein in bacteria. This method was found to be applicable with 0.8, 1.0, and 2.0% agarose gels. DNA fragments varying fro m 23 to 0.4 kb were extracted using this procedure and a yield ranging from 40 to 90% was obtained. The yield was higher for fragments 2.0 kb and high er (70-90%). This range of efficiency was maintained when the starting mate rial was kept between 10 and 300 ng. The heat step was found to be critical since homogenization at room temperature failed to yield any DNA. Extracti ng DNA with our method elicited an increased yield (up to twofold) compared with that extracted with a commercial kit Also, the number of transformant s obtained using the DNA extracted with our method was at least twice that obtained using the DNA extracted with the commercial kit. (C) 2001 Academic Press.