One of the earliest things you probably learned soon after joining your first lab was agarose gel electrophoresis. Pouring and running an agarose gel should be a simple and routine procedure, one that you wouldn’t think could go wrong. In fact, there are a surprising number of ways to destroy your agarose gel. Here are five common culprits of an agarose gel electrophoresis gone wrong:
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 water is used, the gel will melt shortly after applying a charge to the gel box – say goodbye to those samples! (Choosing the right buffer is also important.)
2. Forget to add ethidium bromide
In a rush, it’s easy to forget to add ethidium bromide to your agarose before casting the gel. Without it, of course, it will be impossible to visualize your DNA. Luckily, you can rescue an unstained gel after it’s done running by soaking it in buffer containing ethidium bromide. In fact, some people even prefer to stain it afterward, though you will then have a large volume of ethidium bromide waste to dispose of (luckily, it can be reused a few times before you’ll need to get rid of it).
3. Use the wrong percentage (or type) of agarose
The standard percentage of agarose used to separate DNA is 1.0%. Using more agarose in a gel enhances the resolution of smaller bands, while a lower agarose percentage allows the smaller bands to run through the gel quickly, thus giving you better resolution and separation of larger bands. If the wrong percentage is used, it can be difficult to visualize your bands. Be careful when using a percentage of agarose below 0.8%, as these gels will become weaker 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 to have happen is to accidentally switch the leads from the 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 if you don’t notice the error right away. It’s not a bad habit to check your gel within the first few minutes of starting electophoresis, which also gives you a chance to make sure there’s a current flowing (easily verified by the bubbles coming off the electrodes). If you notice your bands heading in the wrong direction before it’s too late, just switch the leads and run it back the other way.
5. Drop your gel on the way to the imager
Seriously – don’t let this happen to you.
Fortunately, most of us don’t let these things happen to us more than once. What lessons have you learned from your mishaps with agarose gels?