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. The 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 degrees C in a microcentrifuge tube with a Kontes pellet pestle for 1 min. The "homogenate" is then centrifuged for 30 s and the supernatant is saved. The "homogenized" agarose is extracted one more time and the supernatant obtained is combined with the previous supernatant. The DNA extracted using this method lent itself to restriction enzyme analysis, ligation, transformation, and 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 from 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 higher (70-90%). This range of efficiency was maintained when the starting material 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. Extracting DNA with our method elicited an increased yield (up to twofold) compared with that extracted with a commercial kit. Also, the number of transformants obtained using the DNA extracted with our method was at least twice that obtained using the DNA extracted with the commercial kit.
Kurien
B T
BT
Arthritis and Immunology Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, USA. kurienb@omrf.ouhsc.edu
Kaufman
K M
KM
Harley
J B
JB
Scofield
R H
RH
D004247:DNA / Q000032:analysis / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel / Q000379:methods; D015796:HLA-B27 Antigen / Q000235:genetics; D012685:Sepharose / Q000737:chemistry; D013696:Temperature
D015796:HLA-B27 Antigen; D004247:DNA; D012685:Sepharose