Calibration of polyacrylamide gel columns for the separation of oligonucleotides by capillary electrophoresis

Abstract

Polyacrylamide-filled gel columns are used to separate oligonucleotide samples. For homopolymeric standard samples, plots of migration time versus molecular size are presented over a range of 30–160 bases. With 2.5–4 % T and 3.3 % C gels, good resolution over the examined mass range, with peak width at half height of 3 to 6 s, is obtained by applying electrical fields of 200–400 V/cm. The separation of heteropolymeric nucleotides by slab gel electrophoresis under routine conditions was compared with capillary gel electrophoresis. Using the same column and the same separation conditions, the plot of migration time versus base number is linear with and identical slope for three oligonucleotide samples which were examined, allowing a calibration of a gel-filled capillary for molecular mass determination.