Pouring and running an agarose gel is a simple and routine procedure that you probably learned soon after joining your first lab. A procedure that couldn’t possibly go wrong. Or so you’d think. In fact, there are a surprising number of ways to destroy your agarose gel. Here are some of my favorites:
1. Use water instead of buffer for the gel or running buffer
Agarose gels are cast and run using TAE or TBE buffer. Since both of these buffers are clear, it’s easy to mistake them for water. If you do use water though, your gel will melt shortly after applying charge to the gel box – say goodbye to your samples!
2. Forget to add ethidium bromide
In a rush, it’s easy to forget adding ethidium bromide to your agarose before casting the gel. Without it, of course, it will be impossible to visualize your DNA. You can rescue an unstained gel by shaking it in an ethidium bromide solution after it’s done running; but then you have a large volume of waste to dispose of.
3. Use the wrong percentage (or type) of agarose
The standard percentage of agarose used to run a DNA gel is usually around 1.0%. Using a higher agarose percentage enhances resolution of smaller bands; conversely, using a lower agarose percentage allows the smaller bands to run through the gel quickly, giving you better resolution and separation of higher molecular weight bands. If you use the wrong percentage, it can be difficult to visualize your bands reliably. Be careful when you start decreasing the percentage of agarose in your gel, as they become weak and much more prone to breakage.
Low melting point agarose is used for specialized applications, such as in-gel ligation. These gels are very “soupy” and fragile, even at comparable percentages, so make sure you grab the right reagent before mixing up a batch of agarose.
4. Switch the leads from the power source
One of the most frustrating mistakes you can make is to accidentally switch the leads on your power source, so that the gel runs backwards. Since the wells are so close to the end of the gel, you will most likely lose all of your samples before you catch the mistake. If you see your bands heading in the wrong direction, though, just switch the leads and run it back in the other direction.
5. Drop your gel on the way to the imager
Seriously – don’t let this happen to you.
What’s your favorite way to destroy an agarose gel?
I did just that, made my gel in water and forgot to add TAE buffer. Because I read this article immediately after I realized what I had done, I stopped the electrophoresis and yes my gel did melt a little, but thankfully my sample is still in there. Can I even rescue it, using a gel purification kit?
You should be ok with the gel purification kit…… good luck ! :)