What is Pulsed Field Gel Electrophoresis?
Pulsed Field Gel Electrophoresis, or PFGE, is a technique used for the separation of very large DNA molecules.
Under standard electrophoresis conditions, an electric field is applied in one direction on the agarose gel, and DNA molecules larger than 40 kilobases essentially move together through the gel, independently of their size.
In contrast, PFGE applies an electric field that periodically changes direction. By introducing an alternating voltage gradient on a PFGE system, very big DNA fragments can be separated by their reorientation and movement at different speeds through the gel. Larger pieces of DNA are slower to align their charge to the change in direction, while smaller pieces are quicker to align, resulting in greater separation.
Successful PFGE Equipment & Conditions
Let’s take a closer look at the equipment and conditions that will lead to successful PFGE.
Here is the electrophoresis chamber, which contains the running buffer. The agarose gel should be well-anchored in the middle of this chamber, so that it does not float around. There are multiple pairs of electrodes around the gel, set in a hexagonal grid. This hexagonal shape of electrodes allows for the directional switch of the voltage. The voltage is periodically switched among three directions: one that runs through the central axis of the gel, and two that run at an angle of 60 degrees on either side.
The PFGE technique takes longer than standard gel electrophoresis, due to the size of the DNA fragments, and because the fragments do not move in a straight line.
To prevent the agarose gel from overheating due to the extended period under the electrical field, which could result in a loss of resolution, a cooling module adjusts the temperature of the buffer. A typical PFGE program is run at 14 or 15 degrees Celsius. It is important that no air is left in the tubing to and from the cooling module.
On the control module, the migration protocol can be entered and monitored. This includes the initial and final switch times, the duration of the migration, the angle of electrophoresis and the voltage.
The bigger the DNA fragments, the longer the pulse times should be, and the longer the total migration time. The switch time is the length of time the electrical field is pulsed in a single direction. For example, a 10-second switch time means that the electrical field will be pulsed in one direction for 10 seconds and then switched to another direction for 10 seconds.
Samples with a wide range of DNA fragment sizes, such as NEB’s PFG Markers, can be resolved by ramping up from a short switch time to a longer one, over the course of the run.
Most PFGE protocols are optimized for 6 volts per centimeter gel runs and optimized for a 120 degree angle. With any PFGE system, depending on the migration protocol, you can separate DNA fragments or molecules from 10 kilobases to 10 megabases.
Here are separation examples of the Lambda Mono Cut Mix, from 1.5 to 48.5 kilobases, and Yeast Chromosomes, from 225 to 1,900 kilobases.
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