High resolution preparative gel electrophoresis of DNA fragments and plasmid DNA using a continuous elution apparatus.
An apparatus designed for preparative gel electrophoretic separation of proteins (M. A. Hediger, (1984) Anal. Biochem. 142, 445-454) has been used successfully for separating DNA restriction fragments. The apparatus displayed yields and resolutions that are higher than those obtainable with commercially available devices. The amounts of DNA applied to the column range from a few micrograms to milligram quantities. Restriction DNA fragments very similar in size were isolated in pure form with the apparatus. After ethanol precipitation, these fragments were successfully used for restriction enzyme cleavages, ligation, or chemical sequencing. Furthermore the apparatus provides a convenient method for the large-scale isolation of plasmid DNA. The method requires only 4 h of electrophoresis and therefore greatly reduces the preparation time compared with the conventional equilibrium centrifugation method which requires centrifugation times of up to 60 h. In contrast to the centrifugation method, contaminants such as RNA, proteins, and chromosomal DNA are efficiently removed by this technique.
D004247:DNA / Q000302:isolation & purification*; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel / Q000295:instrumentation; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation; D004926:Escherichia coli / Q000235:genetics; D010957:Plasmids; D014461:Ultracentrifugation
D004269:DNA, Bacterial; D004247:DNA; D004262:DNA Restriction Enzymes