Pulsed-field gel electrophoresis

Abstract

DNA molecules longer than 25 kb are poorly resolved by standard agarose gel electrophoresis. These longer molecules can be resolved using several techniques that periodically change the direction of the electric field in the gel. This unit describes the simplest and most generally useful of the pulsed-field techniques, field inversion electrophoresis, which can be tuned to resolve molecules from 10 to 2000 kb (or more with specialized equipment). To resolve molecules beyond the range of field inversion, it is necessary to use some sort of field-angle alternation electrophoresis such as CHEF (contour-clamped homogeneous electric field; described in an Alternate Protocol). A method is also provided for preparing high-molecular-weight DNA samples and size markers embedded in agarose blocks.