How to run DNA and RNA gels at higher voltage (i.e. faster)
If you’ve ever run a DNA or RNA gel at high voltage and cooked/overheated your gel, this post is for you! Scroll to the bottom for the main conclusions of this paper by Sanderson et al which changed the buffer and agarose gel composition to minimize heating. For all my fellow DNA and RNA gel marathon runners: if you want nucleic acid gels to run faster, consider removing EDTA from your buffer! Cover art is the gel from my experiment.
Overheating agarose gel problem
For DNA electrophoresis experiments, the percent agarose is inversely related to nucleic acid size, so smaller percents give you better resolution of larger nucleic acids. I usually look for larger nucleic acids, so I run higher voltage 0.8% agarose gels to lower the amount of time I need to run gels. But what about RNA?
In my opinion, the RNA bleach gel is much easier than pouring a formaldehyde gel. You literally just add clorox bleach to a typical DNA gel (yes, I can and have run DNA on it). However, it still has its own pitfalls, specifically that adding bleach to the agarose mixture significantly increases the heat generated during an electrophoresis experiment, to the point where our myGel mini system spits out an overheating error if you run the gel at 100V for >20 minutes. In our RNA gel protocol, we have to watch the gel closely and switch out the buffer every 10-15 minutes, or risk “losing” our sample (running a gel too hot or too long or causes bands to disappear.)
You could technically run the gel at a lower voltage, but even at 50V for 30 minutes, you produce a lot of heat, which can still cause your RNA bands to disappear. I’ve even tried to run 30V for an hour, but that’s way too long, especially if you are waiting to verify the RNA products before starting an experiment, like we do for oocyte injections.
Note: for a great historical review of DNA electrophoresis, check this paper out!
Solution: Agarose DNA gel without EDTA
I came across this very nice paper that investigates the different properties of the buffer solutions. I highly recommend you read it if you’re interested in running DNA or RNA gels more quickly, or if you’re interested in increasing the resolution between bands. It convinced me that I should run our RNA gels at 100V using 0.5X TB.
My results: if you want to run samples more quickly at higher voltages without worrying about band compression, remove EDTA from your buffer! I ran a DNA gel for 10-15 minutes @ 100V just to see the difference, and the gel wasn’t warm at all. Doing a second run for the same amount of time gave great resolution, and again, was not nearly as warm as a similar experiment in 1X TAE (I would usually have to change out the buffer at least once.)
Testing an RNA bleach gel without EDTA
I also tried running a 0.5X TB RNA bleach gel (0.4 g agarose, 47.5 mL, 0.5X TB, and 2.5 mL of Clorox) and the results were good! I kept my eye on the gel, but you don’t produce as much heat after 15 minutes. I checked the bands under UV, and to my surprise I could see more than just the 3KB ssRNA marker (rare in my experience), so I decided to add another 20 minutes to the run. In the second run, both gels got quite warm, although it was not as hot as what a single 15-minute run with 1X TAE gel would be. One of the RNA gels had the bands become fainter, while the other maintained definition. I suggest you continue monitor the heat level even with a 0.5X TB gel, but RNA bleach gels under these parameters are much more forgiving.
1X TAE RNA gel after about 50 minutes, and 2 buffer changes. Even after changing the buffer, the gel got excessively hot and loses signal.
0.5X TB RNA gel. after about 30 minutes.
Take home from Sanderson et al's paper
To reduce the heat generated during a DNA/RNA gel electrophoresis experiment, lower the gel volume used and minimize the amount of buffer that covers/submerges the gel.
You don’t need EDTA for most DNA gel applications
You can make gels and running buffers using lower than 1X TA or TB buffers
What is interesting: supercoiled (uncut) plasmids migrate slower (i.e. larger than expected) in 1X TB than 1X TBE, and this effect is worse as you lower the strength of the buffer (0.5x, 0.25X, etc).
A useful application for this: running a low-strength gel for DNA gel-extractions of PCR products. The first example that comes to mind is when you’re PCR amplifying/modifying an empty vector for cloning/ligation. Gel-extracting a PCR product from a low-strength gel would theoretically reduce the background in downstream cloning experiments, if your modification was small, like introducing an new restriction site, or adding an epitope/tag.
I’d be interested to see the quality of the DNA after gel extraction from a low-strength gel; yes EDTA chelates Mg2+ to prevent DNA catalysis, but if you’re using distilled-de-ionized water, you shouldn’t have to worry about Mg2+ in the first place. Sounds like an experiment for another day.
The paper analyzes the ideal conditions for supercoiled and linearized DNA, as well as SiRNA, small nucleotides, etc. It’s definitely worth a read!
