Rapid method for purification of plasmid DNA and DNA fragments from DNA linkers using high-performance liquid chromatography on TSK-PW gel》.pdf

Journal of Chromatography, 240 (1982) 155-163 Elsevier ScientificPublishing Company, Amsterdam Printedin The Netherlands CHROM. 14,748 RAPID M E T H O D F O R P U R I F I C A T I O N OF P L A S M I D D N A A N D D N A F R A G M E N T S F R O M D N A L I N K E R S U S I N G H I G H - P E R F O R M A N C E L I Q U I D C H R O M A T O G R A P H Y ON TSK-PW G E L MICHAEL E. HIMMEL* Biotechnology Branch, Solar Energy Research ln~tit!,tp Golden, CO 80401 (U.S.A.) PETER J. PERNA Department of Biochemistry, Colorado State University,Fort Collins, CO 80423 (U.S.A.) and MICHAEL W. McDONELL Molecular Genetics Group,Synergen, Boulder, CO 80301 (U.S.A.) (First receivedNovember 19th, 1981;revised manuscript receivedJanuary 19th, 1982) SUMMARY High-performance size exclusion chromatography (HPSEC) using TSK- G5000 PW in Tris buffer has been found to be a reliable method for the rapid fractionation of D N A ligation products. Plasmid and fragmented phage DNAs were f o u n d t o elute in less than 2 min with recoveries greater than 98 ~. Escherichia coli transfection studies, using plasmid-DNA that had been subjected to HPSEC column fractionation, showed high transformation efficiencies. MgC12, a component of the D N A ligation reaction, was found to produce D N A - c o l u m n support interactions, which resulted in low D N A recoveries. Such interactions were eliminated by che- lation with ethylenediaminetetraacetate prior to chromatography. INTRODUCTION The evolution of recombinant D N A technology has generated a requirement for a separation technique that will resolve from a D N A ligalion reaction, insert DNAs and D N A linkers 1. It is essential to remove the lin