Incomplete Restriction Enzyme Digestion (Restriction Enzyme Digest)

Question

Accepted Answer

Partial digestion observed with both expected fragments and uncut substrate visible on gel; incomplete linearization of plasmid DNA. Common causes: 1) High salt concentration from DNA purification (spin columns) inhibiting enzyme activity 2) Insufficient enzyme units: <3–5 units per µg DNA used 3) Incubation time too short for complete cleavage 4) Enzyme exhibits reduced activity on supercoiled DNA substrates 5) Presence of slow cleavage sites requiring extended incubation 6) DNA contains inhibitors from mini-prep or other purification methods Solutions: 1) For salt-sensitive enzymes (low activity in NEBuffer r3.1), clean up DNA (NEB #T1030) prior to digestion; limit DNA solution to ≤25% of total reaction volume 2) Use ≥5–10 units of enzyme per µg DNA for difficult digests 3) Increase incubation time to 1–2 hours, especially for slow sites or supercoiled substrates 4) For supercoiled DNA, increase enzyme units in reaction 5) Test substrate DNA with control DNA to identify inhibitor presence; clean with spin column (NEB #T1030) or dilute reaction to reduce contaminant concentration 6) Use Monarch Kits (NEB #T1010, #T1020, #T1030) designed to minimize salt carryover