DNA Smearing on Agarose Gel After Digestion

Symptom

Restriction digest product appears as a smear rather than discrete bands on agarose gel, indicating DNA degradation or enzyme-DNA complex formation.

1. 1 Excessive restriction enzyme units causing enzyme to remain bound to DNA substrate
2. 2 Nuclease contamination in running buffer or agarose gel
3. 3 Enzyme-DNA complexes not dissociated before gel loading

1. 1 Reduce enzyme units in reaction to minimum required for complete digestion
2. 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. 3 Use fresh, clean running buffer and freshly prepared agarose gel
4. 4 Clean up DNA using spin column (NEB #T1030) if nuclease contamination suspected

Related Video (2)

Addgene ★ 88

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"Directly demonstrates performing restriction digests and analyzing products on agarose gels, the exact workflow where smearing occurs."

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Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks

"Uses restriction enzyme digestion of plasmid DNA, providing context for proper digest conditions relevant to avoiding over-digestion."