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

A novel high resolution method for the separation of, both, linear and supe rcoiled circular plasmid DNA in the range of 3000-5600 bp is described. Emp loying ultradilute solutions of hydroxyethylcellulose (0.070-0.100% w/w) co ntaining no intercalating agent, we were able to resolve plasmids as well a s 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 po lymer and buffer concentration, capillary coating, salt additives and appli ed voltage has been investigated. The main issue of this study is to explor e the applicability of high performance capillary gel electrophoresis (HPCG E) to molecular cloning and related purposes. Our results demonstrate that this method allows convenient monitoring of cloning procedures. It is super ior to conventional agarose gel electrophoresis with respect to, both, sens itivity and resolution. Hence, it is especially useful for manipulations wh ich result in only a small difference in fragment length or provide only li mited quantities of sample DNA. To summarize, the method described in this paper allows to determine the size, concentration, and purity of plasmid DN A simultaneously in one single step. (C) 2000 John Wiley & Sons, Inc.