Partial Digest with Multiple Uncut Bands

Symptom

Gel shows mixture of fully digested, partially digested, and uncut DNA. Some recognition sites cleaved while others remain intact in the same molecule.

1. 1 Salt inhibition from spin column purification; DNA >25% of reaction volume concentrates salts
2. 2 PCR components (polymerase, dNTPs, DMSO) carried over inhibiting enzyme
3. 3 Incorrect buffer reducing enzyme activity to <50% optimal
4. 4 Insufficient enzyme units (<5 U/μg DNA) or incubation time <1 hour
5. 5 High glycerol concentration from excess enzyme volume

1. 1 Clean up DNA (NEB #T1030) before digestion; use Monarch Kits to minimize salt carryover; DNA ≤25% of reaction volume
2. 2 Purify PCR products with cleanup kit (NEB #T1030) prior to restriction digest
3. 3 Use recommended NEBuffer supplied with enzyme; check buffer compatibility table
4. 4 Use ≥5–10 U enzyme/μg DNA; digest for 1–2 hours minimum
5. 5 Limit enzyme volume to ≤10% of total reaction to keep glycerol <5% v/v

Related Video (3)

Addgene ★ 85

Restriction Digest Analysis

"Directly demonstrates how to analyze restriction digest gel results, which is critical for interpreting partial digest bands."

New England Biolabs ★ 75

Cloning With Restriction Enzymes

"Explains restriction enzyme cloning guidelines, including reaction conditions and enzyme selection, relevant to preventing partial digest issues."

Promega Corporation ★ 60

How to Perform Plasmid Prep

"Shows plasmid prep purification, helping understand how salt carryover could inhibit digestion."