The concentration of agarose in electrophoresis gels was found to have significant effects on the apparent density and release characteristics of the electrophoretic traps for circular DNA. Agarose gels were cast in a range of concentrations from 0.25% to 2.5% (1% is 1g/100 mL). The trapping behaviors of two DNA circles with sizes of 13 kilobase pairs (kbp) and 52.5 kbp were studied. The electric field strength (E) for the onset of trapping, the apparent trap density, and release characteristics of circles from traps were studied using both direct current and field inversion gel electrophoresis (FIGE) experiments. Direct current mobility indicated that the onset of trapping, indicated by a reduction in velocity and increase in band smearing, occurred at significantly lower value of E in 0.25% gels compared to 1% gels or 2% gels. The final value of E for complete immobilization of the 13 kbp open circular form was 16 V/cm in 0.25% gels, and 20 V/cm in 1% and 2% gels. The supercoiled forms were not trapped in agarose gels at the values of E used in this study, but they were used as mobility standards to compare to the open circular forms. FIGE mobility measurements, done by varying the reverse times, were used to determine the time required to free a circle from a trap. At the value of E=3 V/cm, the reverse pulse times required to completely release the 52.5 kbp open circles were 0.1 s in 0.25% gels and 0.18 s in 1% gels. At the value of # = 22 V/cm, the reverse pulse times required to completely release the 13 kbp open circles were 20 ms, 8 ms, and 15 ms from 0.25%, 1%, and 2.5% gels, respectively. The time span of the reverse pulse time (from first release to complete release) was increased in the 0.25% gels compared to the time span for the 1% gels or 2.5% gels. These results indicated that the electrophoretic traps were more heterogeneous in the lower concentration gels. FIGE experiments, in which the forward pulse times were varied, were done to determine an average distance that a circle traveled before trapping occurred. The average distances before the 13 kbp open circles were trapped (E=22 V/cm) were approximately 80 m in 0.25% gels, 180 m in 1% gels, and greater than 500 m in 2.5% gels.
Citation: Separation Science and Technology
Pub Type: Journals
agarose, DNA, electrophoresis, field inversion gel electrophoresis, FIGE, plasmid, separation, trapping