Influence of the agarose matrix in pulsed‐field electrophoresis

Abstract Properties of agarose potentially relevant to PFGE (pulsed‐field gel electrophoresis) are reviewed, and some new information is presented. Agarose polymers appear to have molecular weights in the range of 100 000 to 200 000 Da, but this is not tightly related to the effective gel strenght. Agarose has some residual charge, and hence exhibits electroendosmosis (EEO). It is possible to markedly increase the speed of separation of DNA molecules by using agarose of low EEO, especially in low ionic strenght, non‐borate buffers. This increase is especially noticeable in the relatively long experiments required for separation of large DNAs. It is also possible to increase the range of separation in a single run by use of step gradients of agarose concentration, which allows visualization of yeast chromosomes and lambda‐phage restriction fragments in the same lane. Because of the strong influence of concentration on separation, it may be useful for investigators to control water content and related variables. Our lack of knowledge of the detailed microstructure of gels may be barrier to complete understanding of PFGE.