Rapid isolation of high-molecular-weight DNA from agarose gels.
We have developed a simple, reliable, and rapid method for recovering DNA from agarose gels. While many methods for DNA extraction have already been described, few provide quantitative recovery of large DNA molecules. These procedures generally require costly apparatus, extended elution times, or considerable handling of the sample after elution. Our method employs a novel electroelution chamber constructed from acrylic plastic. Gel slices containing DNA are placed in the chamber between platinum electrodes. Voltage is applied and a continuous flow of buffer sweeps the eluted DNA from the chamber into an external receptacle. Elution is complete in 7 min. Concentrated DNA is obtained by butanol extraction and alcohol precipitation in 1 h. Recoveries, quantitated by counting radiolabeled DNA or by densitometry of analytical gels, were 94 to 100% for fragments of 4 to 50 kb. The eluted DNA was undegraded and could be digested with restriction enzymes, ligated, end-labeled, or used to transform cells as efficiently as noneluted DNA. Complete elution of a 100-kb plasmid, a 194-kb concatemer of bacteriophage lambda, and of 440- and 550- chromosomes of Saccharomyces cerevisiae was also achieved using the same process. This method is suitable for routine use in a wide range of cloning applications, including the electrophoretic isolation of large DNA molecules.
Pollman
M J
MJ
Department of Microbiology, Loma Linda University School of Medicine, California 92350.
Zuccarelli
A J
AJ
D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004587:Electrophoresis, Agar Gel / Q000379:methods; D005782:Gels; D008970:Molecular Weight; D010957:Plasmids; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000378:metabolism; D011549:Pseudomonas / Q000235:genetics; D012685:Sepharose; D014170:Transformation, Genetic
D005782:Gels; D004247:DNA; D012685:Sepharose; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D004262:DNA Restriction Enzymes