Partial Digest with Multiple Uncut Bands (Restriction Enzyme Digest)

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Accepted Answer

Gel shows mixture of fully digested, partially digested, and uncut DNA. Some recognition sites cleaved while others remain intact in the same molecule. Common causes: 1) Salt inhibition from spin column purification; DNA >25% of reaction volume concentrates salts 2) PCR components (polymerase, dNTPs, DMSO) carried over inhibiting enzyme 3) Incorrect buffer reducing enzyme activity to <50% optimal 4) Insufficient enzyme units (<5 U/μg DNA) or incubation time <1 hour 5) High glycerol concentration from excess enzyme volume Solutions: 1) Clean up DNA (NEB #T1030) before digestion; use Monarch Kits to minimize salt carryover; DNA ≤25% of reaction volume 2) Purify PCR products with cleanup kit (NEB #T1030) prior to restriction digest 3) Use recommended NEBuffer supplied with enzyme; check buffer compatibility table 4) Use ≥5–10 U enzyme/μg DNA; digest for 1–2 hours minimum 5) Limit enzyme volume to ≤10% of total reaction to keep glycerol <5% v/v