Large-scale purification of plasmid insert DNA sequences using low-percentage agarose exclusion chromatography
Abstract
Linear double-stranded DNA fragments ranging from 0.14 to 8.4 kbp have been fractionated on lowpercentage agarose exclusion gels. Both Ultragel A2 (2% agarose) and Bio-Gel A150m (1% agarose) exclude DNA fragments > 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 > 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.
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This work was supported by NIH Grant GM11719.