Capillary electrophoresis investigations of pET3aPAI-1 DNA involving optimized restriction digestion, laser-induced fluorescence detection, and micro-preparative separation
Abstract
This work centers around developing methodologies to isolate the PAl-1 coding sequence of the DNA plasmid pET3a-PAl-1. Size Selective Capillary Electrophoresis (SSCE), using entangled polymer filled small i.d. capillaries, is used to develop digestion conditions (time and enzyme concentration) that provide single cuts (at variable positions) of the plasmid using BstY I restriction enzyme. After obtaining optimum partial digest conditions for this enzyme, digestion with Ndel will produce a mixture of fragments that includes the fragment (1354 bp) which contains the intact region of interest. Sensitive detection is achieved via laser induced fluorescence using running buffers containing intercalating dye. Using small i.d. capillary conditions as a starting point, the SSCE system is increased to the micro-preparative scale using various larger i.d. capillaries. The effects of capillary diameter, applied voltage, injection amount, and sample buffer concentration on separation performance are studied. Subsequently, single or limited numbers of injections of the single cut sample using a relatively large i.d. capillary should provide adequate material for digestion with Ndel prior to PCR amplification of the 1354 bp fragment.