A Miniaturized Platform for Rapid Separation of Long DNA Using Pulsed Field Gel Electrophoresis
A. Li, V.M. Ugaz
Texas A&M University, US
Keywords: electrophoresis, DNA, pulsed field, miniaturization
Abstract:We have developed a new miniaturized pulsed field gel electrophoresis apparatus capable of separating large DNA fragments up to 32.5 kb in length. The compact size of the device combined with the use of quartz as the substrate material permit the gel to be continuously illuminated with UV light so that the separation processes can be recorded in real time using a CCD camera. In this way, we are able to perform separations in 1.5 hours that normally require 10 hours or more using conventional benchtop instruments. We investigate a variety of experimental parameters in field inversion gel electrophoresis such as agarose gel type, agarose gel concentration, fluorescent dye, electric field waveform, field strength, and pulse time. We also directly measure fundamental physical parameters associated with DNA migration under these conditions (mobility, resolution and diffusion). In addition to achieving rapid separations, these results provide a previously unavailable rational basis to identify optimal separation conditions and provide an improved understanding of the underlying physical processes governing DNA electrophoresis in pulsed fields.