The Influence Of Agarose Concentration In Gels On The Electrophoretic Trapping Of Circular Dna#

Abstract

The density and structure of the electrophoretic traps for circular DNA in gels were strongly influenced by the concentration of agarose used to form the gels. Agarose gels were cast in a range of concentrations from 0.25% to 2.5% (1% is 1 g/100 mL). The trapping behaviors of two DNA circles with sizes of 13 kilobase pairs (kbp) and 52.5 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 critical value of E for complete immobilization of the 13 open circular form was 16 V/cm in 0.25% gels, and 20 V/cm in 1% and 2% gels. FIGE mobility measurements, done by varying the reverse times, were used to determine the time required to free a circle from a trap. 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 μm in 0.25% gels, 180 μm in 1% gels, and greater than 500 μm in 2.5% gels. The higher trap density and longer trap length in low concentration gels explains the experimental observations that, under some conditions, DNA circles migrate faster in higher concentration gels compared to their migration in lower concentration gels. #Certain commercial equipment, instruments, or materials are identified in this paper to specify adequately the experimental procedures. Such identification does not imply recommendation by NIST, nor does it imply that the materials or equipment are necessarily the best available for the purpose.