Enzyme-DNA Complex Causing Larger Bands (Restriction Enzyme Digest)

Question

Accepted Answer

Larger molecular weight bands than expected appear on gel. Restriction enzyme remains bound to cleaved DNA substrate, retarding migration. Common causes: 1) Excessive enzyme units forming stable enzyme-DNA complexes 2) Loading buffer lacks denaturant to dissociate enzyme from DNA 3) Enzyme dissociation rate slower than gel electrophoresis timescale Solutions: 1) Lower enzyme units in reaction to recommended range (5–10 U/μg DNA) 2) Add 0.1–0.5% SDS to loading buffer to dissociate enzyme-DNA complexes 3) Use NEB Gel Loading Dye, Purple (6X) (NEB #B7024) containing dissociating agents