Capillary electrophoretic separation of DNA fragments under stepwise changes of polymer solutions

We demonstrated techniques of stepwise changes of poly-ethyleneoxide (PEO) solutions for DNA separations in the presence of the electroosmotic flow (EOF). The stepwise changes were performed by injecting different PEO solutions contained in a number of syringes to a polyethylene tube, from which the polymer solutions entered a capillary filled with 1X TBE buffer by the EOF. The separation of DNA markers V and VI was not complete under isocratic conditions, either using 1.0-2.5% PEO solutions containing 0.5 μg/mL ethidium bromide (EtB) or using 2.0% PEO solutions containing 0.1-2.0 μg/mL EtB. Resolution, sensitivity and time of the DNA separation were varied under conditions of either stepwise changes of PEO concentrations from 1.5 to 2.5% in 0.5 μg/mL EtB solutions or stepwise changes of EtB concentrations from 0.1 to 2.0 μg/mL in 2.0% PEO solutions. The optimal separation was obtained under simultaneous stepwise changes of PEO and EtB: 1.5% PEO containing 0.1 μg/mL EtB for 45 s, 1.5% PEO containing 0.5 μg/mL EtB for 120 s, 2.0% PEO containing 0.5 μg/mL EtB for 45 s, 2.0% PEO containing 1.0 μg/mL EtB for 30 s, and 2.5% PEO containing 2.0 μg/mL EtB for the rest. The separation was complete in 17 min and the RSD values of migration times were less than 2.0%.