Larger Than Expected Bands from Enzyme-DNA Binding (Restriction Enzyme Digest)

Question

Accepted Answer

DNA bands migrate slower than expected molecular weight on gel, appearing larger due to restriction enzyme remaining bound to digested DNA fragments. Common causes: 1) Excessive enzyme units causing enzyme-DNA complexes to persist 2) Enzyme not dissociated from substrate DNA before gel electrophoresis Solutions: 1) Reduce enzyme units in reaction to minimum effective amount 2) Add SDS to final concentration 0.1–0.5% in loading buffer to dissociate enzyme from substrate 3) Use Gel Loading Dye Purple (6X) (NEB #B7024) which contains dissociating agents