Large-scale purification of plasmid insert DNA sequences using low-percentage agarose exclusion chromatography.
Linear double-stranded DNA fragments ranging from 0.14 to 8.4 kbp have been fractionated on low-percentage agarose exclusion gels. Both Ultragel A2 (2% agarose) and Bio-Gel A150m (1% agarose) exclude DNA fragments greater than 900 bp, while the exclusion limit of Bio-Gel A50m (2% agarose) is about 350 bp. All gels result in moderate resolution of DNA fragments smaller than the exclusion limits; we generally observe nearly complete one-step separation of fragments that differ in size by a factor of 2. On the basis of these results, we have used these exclusion gels to routinely purify greater than 0.4 mg of plasmid insert DNA sequences in one step and over 2.5 mg with a single column, demonstrating that these gel matrices can be ideally suited for repeated rapid large-scale purification of plasmid inserts. In addition, this knowledge allows for a more rational design of plasmids in those cases where large-scale use of the insert DNA is required.
Department of Biochemistry and Biophysics, Oregon State University, Corvalis 97331.
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JC
Hansen
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Rickett
D002847:Chromatography, Agarose; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004586:Electrophoresis; D010957:Plasmids
D004269:DNA, Bacterial