FAQ: Why do I see a DNA smear on an agarose gel after a restriction digest?

There are several possible reasons why you may see a DNA smear on your agarose gel after a restriction digest. For a discussion on this topic please refer to the video above.

A DNA smear on an agarose gel after a restriction digest can result from one, or more, of the following:  
1. nuclease contamination in the digest reaction
2. issues with the running buffer in the gel box or
3. the restriction enzyme has a high binding afinity to the DNA
The source of nuclease contamination may come from the DNA preparation, the digestion buffer or the water used in the digestion mix.  With proper controls, each of these components may be checked independently after incubation at the designated digestion temperature.  
Running buffers that have been maintained at room temperature for extended periods of time can eventually go off and adversely affect electrophoresis.  If the buffer in the gel box appears cloudy and gel runs show abnormal results, rinse the gel box and use a fresh gel before loading your digestion for best results.
If DNA binding is suspected, adding 0.1 - 0.5% SDS solution after digestion will help the enzyme dissociate from the DNA and allow the appropriate banding pattern to be displayed when run on the gel.
Please refer to the restriction enzyme usage guidelines or troublshooting guide provided for additional information.

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    Restriction Enzyme Digestion Problem: DNA Smear on Agarose Gel

    Learn more about what causes this common problem, and how NEB's enzymes are QC'd to avoid DNA smearing.