Megaseparation Anxiety

Manufactured by Biometra (Germany), the Rotaphor utilizes patented Rotating Field Electrophoresis (ROFE) technology to allow separation beyond the limit of conventional horizontal agarose gel electrophoresis. With this method, an electric field between two primary sets of electrodes is stabilized by two sets of secondary electrodes. The electrodes are positioned within a rotor that is rotated above a stationary gel. This format gives users enormous flexibility by choosing vector angles between

Dec 8, 1997
Shane Beck

Manufactured by Biometra (Germany), the Rotaphor utilizes patented Rotating Field Electrophoresis (ROFE) technology to allow separation beyond the limit of conventional horizontal agarose gel electrophoresis. With this method, an electric field between two primary sets of electrodes is stabilized by two sets of secondary electrodes. The electrodes are positioned within a rotor that is rotated above a stationary gel. This format gives users enormous flexibility by choosing vector angles between 0 and 255 degrees. Additionally, by allowing users to vary rotation intervals between two seconds and 2.75 hours, any DNA between 1 kb and 6 Mb can be separated.

During the course of a run, parameters such as electric field vectors, pulse length, and electric field strength can be changed. Also, a built-in platinum temperature sensor and flow-through valve permit the maintenance of temperatures within the buffer chamber. If the buffer temperature drifts beyond preset limits, the run is stopped until the buffer returns to the proper temperature, at which time the electrophoresis run resumes.

At the heart of the Rotaphor system is the Biometra P25 Power Pack and Rotaphor controller. The Rotaphor controller can store up to 10 user-defined programs that can be linked in any combination. Contained in the manual of the Rotaphor is an extensive, up-to-date library of optimal separation parameters.

Bio-Rad: The CHEF Mapper XA System
Pulsate With Bio-Rad
CHEF-DR II System
CHEF-DR III Variable Angle System
CHEF Mapper XA System
GenePath Strain Typing System
FIGE Mapper Field Inversion System
Bio-Rad (Hercules, Calif.) pulsed into the PFGE market in 1988 with the introduction of the CHEF-DR II System. In the CHEF systems, an electric field is generated by an array of electrodes arranged in a hexagonal contour around the gel and clamped to preset potentials. Adjusting the running conditions allows the CHEF-DR II to resolve DNA fragments from 5 kb to 6 Mb. Users can enhance resolution by entering two sets of running conditions successively. This CHEF-DR II utilizes a fixed 120-degree angle.

The CHEF-DR III Variable Angle System combines patented PACE (Programmable Autonomously Controlled Electrodes) technology with CHEF into one instrument that allows users to set up to three blocks of run conditions. The CHEF-DR III will recall the last set of run conditions, and if interrupted by a power failure, the unit will resume the run at the point where it was interrupted without user intervention. This system allows for separations of DNA ranging from 200 kb to greater than 6 Mb, and users can specify the angle of electrophoresis from 90 to 120 degrees. Users can also select the optimal voltage gradient, switch time, and angle for the specified size range.

The CHEF Mapper XA System from Bio-Rad integrates CHEF, FIGE, and AFIGE (Assymetric Field Inversion Gel Electrophoresis). The built-in algorithm of the CHEF Mapper XA can automatically select the optimum run conditions of a designated separation. With the CHEF Mapper, each vector can be assigned its own voltage and its own switch time (pulse duration). Secondary pulses of defined voltage, duration, angle, and frequency can enhance separation and resolution of very large DNA molecules. Protocols can be refined using the PC Windows¨-based Interactive Algorithm Program, designed for the optimization of separation protocols. The CHEF Mapper XA can store up to 99 simple programs or 20 complex programs with up to 8 blocks of programming each.

The Gene-Path Strain Typing System is optimized for use in clinical laboratories, featuring 22 ready-to-use protocols for commonly encountered organisms and an optional software package for the customization of programs. The Gene-Path unit features flexibility in the range of epidemiological species that can be typed and Bio-Rad offers reagent kits that include all the reagents necessary for screening 50 isolates in duplicate.

The FIGE Mapper Field Inversion System is capable of resolving DNA molecules ranging from 100 to 200,000 base pairs. With the FIGE System, the duration of the forward switch time is approximately three times that of the reverse switch time, resulting in a net forward (downward) migration of DNA through the gel. The FIGE Mapper accommodates both FIGE and AFIGE (where the forward voltage gradient is greater than the reverse) and can generate a nonlinear-shaped switch time ramp to enhance DNA separation. This unit contains 10 stored programs optimized for the separation of DNA ranging from 100 to 150,000 base pairs.

A variety of consumables for pulsed field electrophoresis is available through Bio-Rad, including pulsed field and chromosomal grade agarose, size standards, and DNA plug kits.

Pharmacia Biotech:
The Gene Navigator Pulsed Field System
Navigate With Pharmacia Biotech
Gene Navigator Pulsed Field System
PC 500 Switch Back Pulse Controller
Capable of storing up to nine different run procedures, each containing up to six different phases, the Gene Navigator from Pharmacia Biotech (Piscataway, N.J.) allows for the separation of DNA from 1 kb to 10 Mb by PFGE. Users can choose a Hexagonal Kit or Point Electrode Kit for different gel sizes and positioning of electrodes. The Hexagonal Kit generates straight-line separations of fragments on 15 x 15 cm gels via an array of 20 platinum electrodes. The Point Electrode Kit consists of 8 separate moveable, isolated platinum electrodes for use on 20 x 20 cm gels. With this kit, field geometry can be varied to optimize separation. The built-in cooling coil and buffer circulation pump of the Gene Navigator system assist in maintaining uniform temperature and constant pH.

Pharmacia Biotech:
The PC 500 SwitchBack Conroller
Pharmacia Biotech also offers the PC 500 SwitchBack Pulse Controller, which when connected to most existing power supplies and gel electrophoresis units allows for DNA separation of over 1 Mb via FIGE. The programmable controller permits fixed or ramped pulse times and can store up to 30 single- and 30 multiple-step programs. Pharmacia recommends using the PC 500 with the Hoefer HE 100 SuperSub Submarine Gel Electrophoresis Unit and the MultiTemp III Thermostatic Circulator. Pharmacia Biotech supplies l and yeast DNA size markers and numerous restriction enzymes for pulsed field electrophoresis.

The PPI-200 from MJ Research (Watertown, Mass.) is a programmable power inverter that interfaces with a power supply and a gel box to extend and improve resolution of DNA gels by changing the direction of current flow through the gel for programmed intervals. The PPI-200 maximizes resolution of 15 kb to 2 Mb DNA in any standard gel box and can resolve up to 10 Mb DNA in a dedicated pulse field system. Microprocessor-controlled timing ensures precise switching intervals, and a timer ensures gels are stopped at the proper time. The PPI-200 comes with 12 preprogrammed ramp protocols developed to maximize the resolution of specific ranges of DNA from miniscule 15 kb fragments up to larger yeast chromosomes. The unit comes with Gel TimesTM software, which runs in Microsoft ExcelTM to assist in determining optimal ramp times for resolving specific DNA fragments.

Stay on your toes when shopping for a pulsed field system since these units come with different components. It would be worth the time to consult with the companies beforehand and discuss which system can best suit your laboratory's needs. In the course of writing this article, LabConsumer found representatives from these four companies to be very helpful.

For those running pulsed field gels, LabConsumer investigated a few other companies offering consumables designed specifically for pulsed field gel electrophoresis. Although only four companies supply systems for PFGE, LabConsumer found several more companies that supply agarose, specialized buffer, and molecular weight standards for pulsed field gels.

Not every agarose can handle the rigors of pulsed field separations. Agaroses for PFGE generally have high gel strength, allowing lower concentrations of agarose in electrophoretic gels. A gel matrix with low concentrations of agarose permits more rapid migration of high molecular weight molecules through the gel without a lot of resistance. The Pulsed Field Buffer offered by Life Technologies is formulated for pulsed field applications, and is supplied as a 20X concentrate.

Molecular weight standards for PFGE come in a variety of shapes and sizes. Pipetting can mechanically shear larger DNA molecules. Because of this, standards for PFGE containing large DNA are commonly embedded in low melting point agarose, and users simply insert these plugs into the gel prior to a run. Lambda DNA markers are often supplied as equally spread out or successively larger concantemers, and will generally guide gels up to around 1 Mb. Chromosomal DNA from the yeast Saccharomyces cerevisiae is a popular standard with chromosomes ranging from around 200 kb to 2 Mb. Markers are also available from other yeast strains. The CHEF Genomic DNA Plug Kits from Bio-Rad Laboratories allow users to prepare bacterial, mammalian genomic, or yeast chromosome plugs.

Shane Beck can be reached online at sbeck@the-scientist.com

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