DNA Smearing on Agarose Gel After Digestion (Restriction Enzyme Digest)

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Restriction digest product appears as a smear rather than discrete bands on agarose gel, indicating DNA degradation or enzyme-DNA complex formation. Common causes: 1) Excessive restriction enzyme units causing enzyme to remain bound to DNA substrate 2) Nuclease contamination in running buffer or agarose gel 3) Enzyme-DNA complexes not dissociated before gel loading Solutions: 1) Reduce enzyme units in reaction to minimum required for complete digestion 2) Add SDS to final concentration 0.1–0.5% in loading buffer, or use Gel Loading Dye Purple (6X) (NEB #B7024) to dissociate enzyme from DNA 3) Use fresh, clean running buffer and freshly prepared agarose gel 4) Clean up DNA using spin column (NEB #T1030) if nuclease contamination suspected