You have probably run a standard agarose gel hundreds of times. They are great for visualizing small DNA fragments up to 10 kb, but what if you want to examine really large pieces of DNA or even whole chromosomes?
This is where pulsed-field gel electrophoresis (PFGE) comes in! While the equipment required to run PFGE is much more complicated than the standard agarose gel you are used to, the concept is much the same.
How PFGE Works
Similar to a standard electrophoresis procedure, DNA is pulled through a PFGE gel due to electric charge. In principle, the apparatus itself is essentially the same as a standard electrophoresis unit. Similar to standard electrophoresis, there are electrodes that allow the electric charge to pass through the chamber. In PFGE however, these electrodes surround the gel and are not all active at once. Activating only certain electrodes is what allows the electric current to be modulated at specific angles. While a standard gel is usually only active for a couple hours, PFGE runs can take days. To keep the buffer temperature down, it is run through tubing and through a cooler before being fed back into the chamber.
The arrangement of the electrodes in PFGE is what allows such large DNA fragments to be resolved. As the angle of the electric current is switched back and forth, the DNA is moved side to side allowing larger DNA fragments to move through the gel matrix. The result is an agarose gel that can resolve large fragments of DNA and even intact whole chromosomes at the end of the procedure.
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Personalizing your PFGE Run
Contrary to what you may be used to, a PFGE program is not as simple as setting the voltage and walking away. PFGE power units allow the user to adjust a wide range of the run conditions in addition to voltage, such as the pulse angle and buffer temperature. This customization allows the run program to be adjusted to the specific type of sample being run. Extremely large samples (>MB) will have different run conditions than small ones, and you should adjust the conditions depending on the sample type.
The following components should all be optimized for the best results:
| Component | How to optimize |
|---|---|
| Voltage | The voltage in PFGE is measured as V/cm. I have personally found that most protocols use 6 V/cm as the standard, allowing samples around a few hundred kb to be resolved quite well. If you are working with samples that are much larger in size (megabases), it is generally suggested to lower the voltage. The inverse is true for small fragments. |
| Pulse Angle | The pulse angle is the angle of difference between the electric currents that will be applied. Most protocols use an angle of 120°; however, this can often be adjusted. For instance, use a smaller angle to increase the resolution of large fragments and a larger angle for smaller fragments. Keep in mind, though, that increasing the resolution of one type of fragment often comes at the expense of resolution of the other. |
| Switch Time | Switch time has the largest impact on sample resolution. Switch time refers to how long the current will pull in any one direction. Use a short switch time for small sizes and a long switch time for larger fragments. As you might expect, many samples will contain a wide range of sizes of DNA fragments. To combat this issue, set the program to vary the switch time throughout the run. The program will ramp the switch time from a short time to a longer time over the course of the run to compensate for a variety of sizes within the sample. |
| Temperature | Because the DNA is not moving in a straight line during the run, a PFGE cycle takes much longer to move DNA through the gel matrix. As a result, a single PFGE run usually lasts overnight and sometimes up to a couple of days. These long run times mean that buffer temperature must be maintained throughout the procedure to prevent overheating. This is accomplished by pumping the running buffer through a chiller during the run. In addition, sometimes you need to pause the run and add new buffer if the current buffer becomes exhausted. As you might expect, the run time is impacted by the temperature of the buffer. A lower temperature will mean a longer run time, but often times a greater resolution. Conversely, a higher temperature will result in a shorter run time, but lower resolution. |
Tips and Tricks for PFGE
Running a pulsed-field gel can be exciting. It isn’t often that you get to visualize such large pieces of DNA. However, it can also be extremely frustrating. It isn’t uncommon to wait 24 hours only to find out that your DNA was degraded before you started the run. Then, you have to start all over. What a waste!
Below are some tips that I learned to help take some of the frustration out of PFGE.
1. Be Gentle with Your DNA
The larger the DNA is, the more susceptible it is to shearing. As a result, you have to be extremely careful when you are working with samples for pulsed field gel electrophoresis.
The easiest way to get around this problem is to extract DNA inside of agarose plugs. You can find various protocols around that explain how to do this. I have been happy with those put out by Bio-Rad alongside their gel plug kits. Embedded DNA prevents any shearing forces from acting on it, and makes the DNA much easier to work with. The general protocol involves suspending cells in melted agarose and pipetting agarose and cell mixture into a gel mold before carrying out the DNA extraction. Extraction requires a series of washes to remove cell debris, followed by a restriction digestion as a wash as well. Finally, load the agarose plugs into the pulsed field gel for electrophoresis.
If your DNA is already in a liquid form, be extra careful. Stay away from any procedures that require you to vortex your DNA, and try to be gentle. When pipetting, be sure to use wide-bore pipette tips to reduce the forces on the DNA. You can purchase wide-bore tips from a supplier, but I just use a razor blade to cut the end off of standard pipette tips.
2. Choose Agarose Wisely
While technically you can use standard agarose for pulsed field gel electrophoresis, in general, the quality is much lower. In my attempts to use standard agarose, I observed that the DNA does move through the gel but the bands became diffuse. This made it difficult to compare to a size standard. Luckily, companies have created agarose specifically for pulsed field applications. While they won’t say exactly what is different between the different options, most agarose options for this purpose have a high tensile strength and a low amount of negatively charged contaminants (EEOs). The end result is a gel that gives a higher resolution than the standard options. In my experience, however, these gels do tend to fracture more easily during handling. Always move the gel in a tray to prevent it from tearing.
3. Clean the Pulsed Field Gel Electrophoresis System Regularly
The pulsed field system is a complicated network of tubes, reservoirs, and chilling units. The result of this complexity is a system that easily becomes a breeding ground for bacteria. Other than being gross, contaminated pulsed field units will also produce poor results.
To prevent your system from growing unwanted organisms, fully drain the solution from the system in between runs, especially from the tubing connecting the various components. Additionally, drain the running solution from the system and run distilled water through it before cleaning it out completely. I usually turn the pump on and run the unit until all of the solution has been pumped out before stopping for the day.
If you are concerned that the system has become contaminated, you can safely pump a 50% bleach solution through the system to remove any contaminants. Just make sure to do a few distilled water washes before using it again.
4. Pay Attention to the Buffer
When running gels for a long period of time, it is important not to exhaust the electrophoresis solution. In my experience, the best way to do this is to use TBE buffer for all pulsed field gels. You may use TAE for standard gels, however, the buffer will not generally stand up to the longer conditions required in a pulsed field gel.
Also, add fresh buffer every 12-15 hours. Nothing is worse than coming into the lab in the morning and finding that your 24 hour pulsed field gel has stopped partway through the night due to the buffer strength giving out. To prevent this, I generally exchange about 50% of the buffer every 12-15 hours to be safe.
Final Thoughts
While pulsed field gel electrophoresis may seem complicated at first, remember that it is not too different than standard electrophoresis. Like many of the procedures you face in the lab, the best results will come after some trial and error.
Luckily, you can find many published protocols containing run conditions for a variety of different sample types. While that is a good place to start, don’t be afraid to modify the settings for your needs. You should be well on your way to beautiful high molecular weight separation! Good luck!
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