Every research lab has its own way of doing things, and some are so standard that not much attention is paid to them. This is certainly the case for agarose gels.
Agarose gels are a multipurpose tool in a biological laboratory; you use them to check plasmid preparations before DNA extraction, PCR results, new clones… There are so many uses for agarose gels that you often don’t think much about the best way to run them.
But when your gel’s image doesn’t look pretty, you will wish you had taken those extra five minutes before you started and thought it through more thoroughly.
Been there, done that. Here are a few starting points for things to think about (preferably before you start, but also if you end up with horrible gel pictures and can’t figure out why) to get picture-perfect gels every time.
Top Tips for Troubleshooting Agarose Gels
TBE or Not TBE: Choosing the Right Buffer
The most popular buffers are TAE (Tris-Acetate-EDTA) and TBE (Tris-Borate-EDTA). You need the buffer both to prepare the gel and to fill the chamber in which the gel will run.
In general, labs tend to prefer one over the other, but it is good to know the differences so you can make the appropriate choice for your specific gel run. A few points to consider when selecting a buffer include:
TAE is best suited for larger DNA fragments, while TBE works best for smaller DNA fragments as it provides greater resolution.
TBE has greater buffering capacity and is recommended for longer gel runs; however, the borate in TBE is an enzymatic inhibitor, so if you plan to use the DNA for any downstream application TAE is the way to go.
stocks of TAE are more stable over time, while TBE stocks can precipitate in a few weeks (Tip: I usually use TBE for my assays, so I make sure to prepare a small amount of 5X stock to avoid precipitation).
Make sure you consider these differences when choosing the best buffer for your assay’s purpose.
Keep It Fresh (the Buffer That Is…)
After you have decided which buffer will work best for your experiment, you should ALWAYS have a fresh aliquot to prepare your gel with and run it in.
By fresh, I mean freshly diluted buffer that hasn’t been used before. It is okay to keep a stock of 5X or 10X buffer (or higher in the case of TAE), but make sure to dilute it right before use.
Do not reuse buffer from previous runs – the time you save from not making up fresh buffer will be negligible compared with having to run the whole experiment again to get a usable picture. When I started only using fresh buffer, I started getting sharper, more even bands throughout my samples. Yay!
Let the Gel Take Its Time
This is the most important tip: don’t rush an agarose gel! You don’t want your beautiful cloning result to be ruined by a terrible picture just because you were in a hurry.
You have to run your gel at under 75V and make sure the buffer is not overheating. You can reduce the risk of overheating by cooling down the buffer and/or the gel before the run, or run the gel in a cold room.
Be careful with large gels: they may get warm in the center while the edges are cool. One of the main causes of smeared, uneven bands is the temperature of the buffer. You can (and should) avoid that by maintaining a constant voltage of around 50V to 75V.
Presentation Matters
Remember that your result is only as good as the way you present it. There is no way to convince your boss that a bad gel picture will be sufficient just because the fragment “is there”. Don’t waste the time it took you to actually get the data by rushing the gel run step!
Do you have any tips for troubleshooting agarose gels? Share them with us in the comments!
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