High performance capillary gel electrophoresis as a method to separate plasmid-DNA cloning vectors with very high resolution (below 100 bp) and its application in molecular biology

Abstract

A novel high resolution method for the separation of, both, linear and supercoiled circular plasmid DNA in the range of 3000–5600 bp is described. Employing ultradilute solutions of hydroxyethylcellulose (0.070–0.100% w/w) containing no intercalating agent, we were able to resolve plasmids as well as linear fragments with a size difference of about 100 to 55 bp (R=0.5), depending on their size, at a limit-of-detection of about 3 ng (UV/VIS 260 nm, absolute amount). The effect on the separation efficiency of varying polymer and buffer concentration, capillary coating, salt additives and applied voltage has been investigated. The main issue of this study is to explore the applicability of high performance capillary gel electrophoresis (HPCGE) to molecular cloning and related purposes. Our results demonstrate that this method allows convenient monitoring of cloning procedures. It is superior to conventional agarose gel electrophoresis with respect to, both, sensitivity and resolution. Hence, it is especially useful for manipulations which result in only a small difference in fragment length or provide only limited quantities of sample DNA. To summarize, the method described in this paper allows to determine the size, concentration, and purity of plasmid DNA simultaneously in one single step. ©2000 John Wiley & Sons, Inc. J Micro Sep 12: 75–86, 2000