Troubleshooting issues with DNA ladders begins with determining the issue, such as smeared bands, faint bands or missing bands. Next, determine the root cause of the issue: contamination, forgetting to add your ladder, using the wrong buffer or not loading enough of your ladder in the gel.

In some cases, you will have to run your gel over again, but you should have a lot more success after diagnosing the issue.

So in this article, we’re explaining some DNA ladder issues and how to solve them.

Article Table of Contents

Why is my DNA ladder smearing?

Why is the DNA ladder not separating?

Why is my DNA ladder faint or missing?

Ready-to-use DNA Ladders

GoldBio sources

GoldBio Related Products

Keywords

References



Why is my DNA ladder smearing?

The possible reasons a DNA ladder smears include degradation of the DNA, too much of the ladder loaded onto the gel, DNA contaminated with proteins, or inadequate gel running conditions.

Generally, DNA ladder fragments appear as clear bands in the gel and some minimal smearing is tolerable. However, when you encounter thick, highly pronounced smears in your bands, you will need to redo the gel and use a fresh DNA ladder.

If your DNA ladder is smeared, you can try the following solutions:

Degrading Ladder:

degrading DNA Ladder band illustration

DNA ladder bands with degradation will look like a thin band with a short smeared tail.

Carefully handle the DNA ladder and use DNase-free pipette tips with a filter to avoid contamination with DNases that can degrade DNA.





Excessive Load:

DNA ladder band smear due to excessive load - illusration

DNA ladder bands smearing due to too loading too much will usually have a wider band with a stronger smeared tail.

Load the recommended amount of DNA on the gel. This amount varies depending on the manufacturer. Use between 3-5 μl/well for GoldBio ladders (0.5 μg).




Protein Contamination:

DNA Ladder band with protein contamination - Illustration


DNA ladder bands with protein contamination will have a wider, brighter band with a strong, smeared tail.

The DNA contaminated with proteins generally run as a high molecular band.Redo your gel with a fresh DNA ladder in this situation.




Why is the DNA ladder not separating?

Your DNA ladder may not be separated due to inadequate running conditions. Remember that the rate of migration of DNA molecules on a gel is determined by the size of DNA, the agarose concentration, the voltage applied, the type of agarose, and the buffer (Lee et al. 2012).

If you’re encountering little to no separation, one of the first things you should do is compare the size of your fragment and ladder to the run time you’ve given it to make sure you’ve allowed the appropriate time for electrophoresis.

Next, you’ll want to look at your other conditions, the voltage applied, the agarose concentration, and consider areas that might need better optimization.

Furthermore, let’s picture a situation where in one lane you have two DNA fragment bands, and there is separation between them, but the ladder itself is a streaky blob.

In that case, it is possible your DNA ladder is contaminated with DNA binding proteins used in cloning such as restriction enzymes or ligases. These proteins affect the migration pattern of your DNA ladder because they make it heavier, altering the migration pattern.

You can fix this by using fresh or new DNA ladders for your gel and check if the ladder is correctly separated.

Possible causes for low or no ladder separation

  • Inadequate agarose concentration for the DNA sizes.
  • Inadequate power supply.
  • Inappropriate type of agarose.
  • Use of a different buffer in the gel compared to the buffer used for the DNA ladder.
  • Denaturation


Troubleshooting DNA Ladders with low or no band separation

  • Keep in mind the relationship between the agarose concentration and the range of efficient separation for different DNA sizes. Most gel concentrations range between 0.5% - 2%.
  • Use a power supply of 1-5V/cm between electrodes.
  • Use traditional agarose to separate DNA fragments. Use polyacrylamide to separate DNA fragments smaller than 100 bp.
  • Use a buffer volume lower than 1/3 of the capacity of the flask.
  • Use the same buffer to run the gel as you used in the DNA ladder. The most common gel running buffers are TAE and TBE.
  • Do not heat the DNA ladders before running the gel. Keep the temperature below 30°C during electrophoresis.
  • The pH can also denature DNA. A pH of 8.0 is recommended. However, this can change depending on the manufacturer's recommendations.


Table of appropriate agarose concentrations:

Concentration of Agarose (%)

DNA size resolution (bp)

0.5

1,000 – 25,000


0.75

800 – 12,000


1.0

500 – 10,000


1.2

400 – 7,500


1.5

200 – 3,000


2.0

50 – 1,500


Note: Check our Agarose Gel FAQs to learn more about agarose gels.



Why is my DNA ladder faint or missing?

DNA ladders can be faint or missing because there is a low amount of DNA ladder loaded into the well, it was denatured or degraded, or the DNA ladder may have run off the gel. Also, some poorly resolved bands can end up merged with other bands.

First, a faint DNA ladder is a sign you at least have the ladder on your gel but the load or concentration was not enough when preparing the gel.

A completely missing DNA ladder could be an indication of either forgetting to load the ladder or the DNA ran off the gel.

Just to clarify, a ladder that ran off the gel means the gel run time was too long, and fragments migrated off the gel.

A sign your DNA fragments were run off the gel is when you may find a streaky blob in the top of the gel but no bands. That could mean your DNA fragments are probably in the buffer solution. In other cases, you find bands at the very extreme of the gel or there is an accumulation of bands in that border. In either case, decreasing the run time should fix this issue.

Missing DNA Ladder:

missing DNA ladder how it would look - illustration


A missing DNA ladder may indicate you forgot to load it. A good practice is to load the DNA ladder at the end after the samples and always have a checklist when you are preparing your electrophoresis.

Verify you loaded the DNA ladder. To do this, designate a well to load the ladder into and verify it is full when you finish loading it. A good indication is that the well will be stained with the ladder’s dye. The loading dye present in the DNA ladder can be blue, purple or red.

The appearance of a missing ladder (empty lane where the ladder should be) could also mean the ladder was degraded. Use DNase-free pipette tips when loading your gel.

Your gel run time might have been excessive and the DNA ladder ran off the gel. Reduce the running time to avoid the DNA ladder and samples running off.

Faint DNA Ladder:

faint DNA ladder bands in a gel electrophoresis - illustration


If bands appear faint, increase the amount of loaded DNA. For GoldBio ladders, we recommend loading the gel with 3-5 μl/well (0.5 μg).

To avoid denaturation, do not heat the DNA ladder.





Ready-to-use DNA Ladders

At GoldBio, we recommend using our ready-to-use DNA ladders. The advantage is you don’t need to worry about preparation because our DNA ladders already include the loading dye (e.g., Orange G, xylene cyanol FF).

Keep in mind that a loading dye used in gel electrophoresis has three primary goals:

  • Loading dyes add density to the sample, allowing it to sink into the gel.
  • Loading dyes provide color to identify the DNA fragments and simplify the loading process.
  • Loading dyes move at standard rates through the gel, estimating the distance that DNA fragments have migrated.

My general advice is to work with ready-to-use DNA ladders to simplify the electrophoresis process.



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Keywords

DNA ladder, troubleshooting, tips with DNA ladders.




References

Lee, P. Y., Costumbrado, J., Hsu, C. Y., & Kim, Y. H. (2012). Agarose gel electrophoresis for the separation of DNA fragments. Journal of Visualized Experiments, 62, 1–5. https://doi.org/10.3791/3923

Singhal, H., Ren, Y. R., & Kern, S. E. (2010). Improved DNA electrophoresis in conditions favoring polyborates and lewis acid complexation. PLoS ONE, 5(6). https://doi.org/10.1371/journal.pone.0011318