Pulsed field gel electrophoresis using a double-decker gel system.

Pulsed field gel electrophoresis (PFGE) is capable of resolving large DNA molecules of up to two million nucleotides and is becoming a widely used technique in DNA studies (1—3). Three factors can potentially limit PFGE in a laboratory: 1. the cost of the apparatus, 2. reproducibility of electrophoretic conditions, and 3. unusually long electrophoretic time. To alleviate some of these problems we have tested the simultaneous electrophoresis of two gels in a double-decker setup using a Bio Rad Chef-Dr II apparatus. By employing a buffer flow rate of 0.5 l/min the upper gel is stabilized in a completely flat and horizontal position supported on top of the slightly protruding comers and edges of the bottom gel (Fig. 1). Subsequent Southern hybridization showed a complete lack of cross contamination between the two gels (Fig. 2). Under the electrophoretic conditions used in Fig. 2, running double-decker gels resulted in a 6% reduction in the distance travelled by the DNA in comparison to the use of a single gel. We have noted that the two gels in the double-decker gave identical DNA resolution (Fig. 2), making it feasible to perform accurate linkage mapping of adjacent genetic loci by overlaying the two resulting Southern autoradiograms and searching for common bands (unpublished results). This contrasts with a 5% difference between gels electrophoresed under the same conditions in two separate sets of Chef-Dr II apparatus. Placing a dialysis membrane (not routinely used by us) between the gels had no effect, whereas a sheet of plastic (overhead transparency) deformed the gel tracks in the upper gel significantly (data not shown). The described double-decker procedure therefore offers the advantage of doubling our PFGE capacity, and provides us with gels that can be directly compared to each other.