THE EFFECT OF COLUMN LENGTH, APPLIED VOLTAGE, GEL TYPE, AND CONCENTRATION ON THE CAPILLARY ELECTROPHORESIS SEPARATION OF DNA FRAGMENTS AND POLYMERASE CHAIN-REACTION PRODUCTS

This work examines the effect of different parameters on migration tim e, resolution, and speed of analysis of DNA fragments and PCR products . These parameters include column length, applied voltage, gel type an d concentration, and buffer ionic strength. Our results indicate that 1 cm capillary at an applied voltage of 185 V/cm, filled with commerci al gel, was adequate for the separation of small DNA fragments in unde r 1 min. Resolution of large fragments is directly proportional to col umn length at the same field strength. Also, resolution of large fragm ents is higher (better) at lower field strength at constant column len gth. Analysis is fastest (high throughput) using a short capillary and moderate field strength (200 v/cm). CE using a single short capillary (2-7 cm) is comparable to slab gel in throughput, but more economical . The Sigma DNA buffer and hydroxyethyl cellulose liquid gel gave equi valent results in terms of resolution and reproducibility. The Sigma D NA replaceable gel gave reproducible results when used as received or diluted at 60%. In our hands hydroxyethyl cellulose gave more reproduc ible results than polyacrylamide gel.