GelRed® and GelGreen® troubleshooting
Many customers use GelRed® or GelGreen® precast gels for convenience. However, because GelRed® and GelGreen® are high affinity dyes designed to be larger dyes to improve their safety, they can affect the migration of DNA in precast gels. Some samples, such as restriction digested DNA may migrate abnormally in GelRed® or GelGreen® precast gels.
Tip #1: Load less DNA
Smearing and smiling in GelRed® or GelGreen® precast gels most often caused by overloading of DNA. If you see band migration shifts or smearing and smiling, try reducing the amount of DNA loaded. The recommended loading amount for ladders and samples of known concentration is 50-200 ng/lane. For samples of unknown concentration, try loading one half or one third of the usual amount of DNA. This usually solves band migration problems.
Tip #2: Try the post-staining protocol
To avoid any interference the dye may have on DNA migration, we recommend using the post-staining protocol. If your application requires loading more than the recommended amount of DNA, use the post-staining protocol. While we recommend post-staining gels for 30 minutes, you may be able see bands in as little as five minutes, depending on how much DNA is present. Post-staining solutions can be reused. See the GelRed® Product Information Sheet or GelGreen® Product Information Sheet for detailed protocols.
Other tips to improve agarose gel resolution:
- If you see DNA migration issues or smearing after post-staining with GelRed® or GelGreen®, then the problem is not caused by the nucleic acid dye. Avoid overfilling gel wells to prevent smearing of DNA down the surface of the gel.
- Pour a lower percentage agarose gel. Higher molecular weight DNA separates better with a lower percentage gel.
- Change the running buffer. TBE buffer has a higher buffering capacity than TAE buffer.
There are a few possibilities:
- The dye may have precipitated out of solution.
- Heat the GelRed® or GelGreen® solution to 45-50°C for two minutes and vortex to dissolve.
- Store dye at room temperature to avoid precipitation.
- If you are seeing high background staining of the gel, the agarose that you are using may be of low quality. Contaminants in the agarose may bind to the dye, resulting in increased background.