Bio-Rad CHEF Mapper® XA System User Manual

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Section 1
Introduction

1.1 Overview

Pulsed field electrophoresis is a powerful technique for resolving chromosomal sized

DNAs.

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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 is based on two leading technologies, CHEF (clamped homogeneous

electric fields)

and PACE (programmed autonomously controlled electrodes).

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.

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.

1.2 CHEF Mapper System Specifications

Algorithm

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.