Incomplete Restriction Enzyme Digestion (Restriction Enzyme Digest)

Question

Accepted Answer

DNA substrate is not fully cleaved after restriction enzyme incubation. Uncut or partially cut DNA bands persist on agarose gel alongside expected digestion products. Common causes: 1) Recognition site blocked by Dam, Dcm methylation (bacterial DNA) or CpG methylation (eukaryotic DNA) 2) Salt inhibition from DNA purification spin columns; DNA solution >25% of total reaction volume 3) PCR component carryover (polymerase, dNTPs, primers) inhibiting enzyme activity 4) Insufficient enzyme units (<3–5 U/μg DNA) or incubation time too short 5) Supercoiled plasmid DNA reducing enzyme activity on topology-sensitive enzymes 6) Presence of slow sites requiring extended incubation (1–2 hours) 7) Contaminants in miniprep DNA inhibiting enzyme catalysis Solutions: 1) Check methylation sensitivity; grow plasmid in dam⁻/dcm⁻ strain (NEB #C2925) if blocked by bacterial methylation 2) Clean up DNA with spin column (NEB #T1030) to remove salt and inhibitors; ensure DNA ≤25% of reaction volume 3) Purify PCR products prior to digestion using cleanup kit (NEB #T1030) 4) Use ≥5–10 U enzyme/μg DNA; extend incubation to 1–2 hours for complete digestion 5) Increase enzyme units 2–5× when digesting supercoiled plasmid DNA 6) Use recommended NEBuffer supplied with the enzyme; verify buffer compatibility 7) Assay with control DNA spiked into reaction to confirm inhibitor presence