A new sieving matrix consisting of a mixture of two commercial polymers with different chemical structures, PVP (Mw=1,000,000 g/mol) and hydroxyethyl cellulose (HEC, Mw=250,000 g/mol), has been successfully used for single-stranded DNA separation. This sieving matrix was optimized with a Doehlert design, a second-order model statistical design. A total concentration of 3.5% of polymer, which contains 20.4% of PVP relative to HEC, combines the superior coating capability of the PVP with the high efficiency of the HEC. The use of this new sieving matrix gives highly resolved and reproducible separations of single-stranded DNA ranging from 50 to 500 bp. The separation of these DNA fragments is accomplished in less than 30 min under 319 V/cm with efficiencies up to 4 million plates per meter (measured at 350 bp). More than 90 successive analyses were achieved without any deterioration of the separation performance. Repeatability values of resolution (given as %RSD) for the analyzed DNA fragments are 2.2% (measured with the couple 139/150 bp) and 5.3% (measured with the couple 490/500 bp). Moreover, this mixture has a low viscosity (388 cP), which permits easy filling of fused-silica capillaries. This new sieving matrix, which exhibits high sieving performance, good dynamic coating ability, and low viscosity, can be a useful alternative to other less easily synthesized sieving matrices and eliminates the need for precoating the capillary to eliminate electroosmosis.