DNA Smear on Agarose Gel (Restriction Enzyme Digest)

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Accepted Answer

Digested DNA appears as a smear rather than discrete bands on agarose gel. No sharp bands are visible across a wide molecular weight range. Common causes: 1) Restriction enzyme remains bound to substrate DNA after digestion 2) Nuclease contamination in running buffer or agarose gel 3) Excessive enzyme units causing non-specific DNA degradation 4) Contaminated DNA sample with endogenous nucleases Solutions: 1) Add 0.1–0.5% SDS to loading buffer to dissociate enzyme from DNA, or use NEB Gel Loading Dye, Purple (6X) (NEB #B7024) 2) Use fresh, clean running buffer and freshly prepared agarose gel 3) Reduce enzyme units in the reaction to recommended level (5–10 U/μg DNA) 4) Clean up DNA sample with spin column (NEB #T1030) before gel loading