There can be a few different reasons why you observe additional bands in your digest. For a discussion on this topic please refer to the video above.
Typically, off-target DNA bands are caused by either partial digestion or Star Activity. You need to compare your digestion to the expected DNA banding pattern. If the bands in both lanes are similar to the expected pattern and the additional bands are limited to spaces within the upper and lower bands of the expected pattern, the digestion is incomplete (partial). In this case, you may need to purify the DNA to remove any contaminants, use more enzyme and/or increase the incubation time to ensure complete digestion.
If the additional bands are also seen below the lowest band of the expected pattern, and expected bands are being cut, the digestion is likely to be exhibiting Star activity. Your reactions conditions are driving the enzyme to cleave additional, non-canonical DNA sequences. You should repeat the digestion under one, or more, of the following modified reactions conditions:
∙ Decrease the amount of enzyme
∙ Decrease the incubation time
∙ Increase the reaction volume
∙ Use an appropriate NEB High Fidelity (HFTM) restriction enzyme