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5 ways to destroy your agarose gel

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?

7 Comments

  1. Mo on October 16, 2017 at 4:52 am

    Drop your gel on the way to the imager can be fixed. I have tried. And I was having a great puzzle time.

  2. parichehr on June 20, 2017 at 4:39 pm

    HI

    I have a strange problem with electrophoresis , band lanes disappear on agarose gel near the wells during the electrophoresis, it seems they stuck there and disappear and finally I see a clean gel without any dye and trace of bands.
    I have changed powers ,tanks and buffers. I suppose that some thing goes wrong with agarose. but this pack of agarose powder was ok couple of monthes ago, any help?
    thanks

    • Dr Amanda Welch on June 22, 2017 at 12:13 pm

      My first thought would be the loading dye. When I’ve had that problem, my loading dye was contaminated with some sort of protein. Have you switched that out?

  3. Pat Arbour-Reily on February 17, 2017 at 5:23 pm

    I was called back to the gel area by a panicked student worker …I viewed his gel, melted from the middle, outward, steam rising ( all borders of the gel still gelled…but not for long). A new experience for me in spite of having run gels for almost 4 decades.) I checked electrical parameters of the run , on the power supply. Yup, a problem. Apparently , the gel was made correctly but the running buffer used was the 5X stock . Student had neglected to dilute, as per written instructions ( AND, of course, hands on demos , when he was trained)…..He hadn’t bothered to check the voltage nor amperage ,either , when starting the run, also in the instructions…I guess he felt that was a waste of a precious 3 seconds of time ?
    I thought, SURELY, that I had made every mistake humanly possible, in my decades in the lab….I was wrong. LOL! A learning experience for myself and the student involved.

  4. Katherine on January 5, 2017 at 8:47 pm

    If you realize partway through running that you forgot the ethidum bromide, you can also just add it to the bottom of the gel. Thanks to its charge it will run the opposite direction of your samples. It won’t look pretty, but you’ll be able to see your samples.

  5. Tzar-ina on May 16, 2013 at 10:39 am

    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?

    • Nick Oswald on May 16, 2013 at 3:20 pm

      You should be ok with the gel purification kit…… good luck ! 🙂

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