Pulsed field electrophoresis is a powerful technique for resolving chromosomal sized DNAs.153 Alternating the electric field between spatially distinct pairs of electrodes causes megabase (mb) size DNAs to re-orient and move at different speeds through the pores in an agarose gel. The CHEF Mapper system separates large and small DNA fragments with bet- ter resolution, speed, and accuracy than traditional pulsed field methods. DNAs ranging from 100 bases to over 10 megabases may be effectively resolved. For example, the chromosomal DNA of Schizosaccharomyces pombe can be resolved in 1 day using a 106° pulse angle, com- pared to 2 days at 120°. Everything from Yeast Artificial Chromosomes (YACs) to M13 inserts can be separated with a single instrument. Applications include top down and bottom up mapping (Not I and cosmid cloning, respectively), electrophoretic karyotyping, analysis of tumor cell DNA rearrangements, DNA damage and repair, mammalian DNA analysis, sep- aration of linear and circular DNAs, separation of large proteins, and analysis of bacterial, yeast, and parasite strain homogeneity. The CHEF Mapper system offers innovations beyond original PACE. For example, nonlinear switch time ramping allows linear separations for many sizes of DNA. Secondary pulses, or interrupts, unhinge DNAs from obstructions and permit faster separations. The CHEF Mapper system contains 5 years of protocols embedded on a microchip, eliminating trial and error in setting parameters. Given the size range you expect to separate, the embed- ded auto algorithm interrelates the sizes with 10 other variables, and provides the preferred operating conditions. Common gel and buffer conditions, and run temperature of 14 °C, are assumed. An Interactive Program Disc provides an extended version of the algorithm. This PC program allows you to vary gel, buffer, and temperature parameters and print out optimal conditions. The CHEF Mapper system includes a number of other advanced features, as out- lined in the next sections. Overview articles and specific applications are listed in Section 12. The CHEF Mapper system is based on two leading technologies, CHEF (clamped homogeneous electric fields)31 and PACE (programmed autonomously controlled electrodes).32 The system pro- vides highly uniform, or homogeneous, electric fields within the gel, using an array of 24 electrodes, some of which are clamped, or held to intermediate potentials to eliminate lane distortion. Thus, lanes are straight. The system maintains uniform fields using patented Dynamic Regulation (US patent num- ber 4,878,008). The electrodes sense changes in buffer conductivity due to buffer breakdown, buffer type, gel thickness, pH fluctuations, and temperature, and potentials are readjusted immediately to maintain uniform fields, thus insuring high resolution. In PACE, each electrode’s voltage is controlled independently by firmware. Whereas other CHEF systems are limited to two vectors and a 120° pulse angle, the CHEF Mapper system allows up to 15 vectors per block with a total of 8 blocks, each vec- tor with its own voltage, angle, and duration. Thus, the CHEF Mapper system may simulate virtually any pulsed field technique using homogeneous fields, including FIGE, CHEF, and RFGE. Advanced programmers may simulate OFAGE and other non-homogeneous field methods using a computer. Embedded algorithm for automated optimization of common electrophoresis conditions: Enter smallest and largest size DNA expected in the sample (range 1 kb to 6 mb). Smallest fragment is placed approximately 9 cm from the well. Algorithm assumes 1% PFC agarose, 0.5x TBE buffer, 14 °C for DNAs less than 2.5 mb. For DNAs over 2.5 mb, 0.8% PFC agarose, 1.0x TAE, and 14 °C are assumed. Interactive computer algorithm for full optimization of electrophoresis conditions requires PC 80386 or compatible, with Microsoft Windows® 3.1. Buffer type, buffer concentration, agarose concentration and type, and buffer temperature, can be varied as inputs. The power module contains the electronics for pulsed field electrophoresis, including a 350 V power supply, the switching functions, and drivers for the 24 electrodes. The front panel contains a two line fluorescent display, buttons, switches, jacks, and a fuse as described in Section 4. The fused power supply operates with a maximum of 9 V/cm, or 350 V. The lowest gradient is 0.6 V/cm, or 20 V. The drivers provide clamped homogeneous electric fields in the chamber and maintain them regardless of the pulse angle selected. This feature, dynamic regulation, regulates the potentials so that the proper voltages are maintained regardless of gel size or fluctuations in buffer conductivity or temperature.