Restriction Enzyme Complete Failure to Cleave DNA (Restriction Enzyme Digest)

Question

Accepted Answer

No cleavage observed in restriction digest reaction; DNA remains uncut on gel, resulting in few or no transformants in downstream cloning applications. Common causes: 1) Recognition sequence is blocked by methylation (Dam, Dcm, or CpG methylation depending on DNA source) 2) Incorrect reaction buffer used instead of enzyme-recommended buffer 3) DNA contaminated with inhibitors from purification procedure 4) Insufficient nucleotide spacing: <6 nucleotides between recognition site and DNA end in PCR fragments 5) PCR components (polymerase, dNTPs, primers) directly inhibit enzyme activity Solutions: 1) Check methylation sensitivity of enzyme; if Dam/Dcm-sensitive, grow plasmid in dam⁻/dcm⁻ strain (NEB #C2925) 2) Use the manufacturer-recommended buffer supplied with the restriction enzyme 3) Clean up DNA using spin column purification (NEB #T1030) to remove contaminants 4) Design PCR primers with ≥6 nucleotides between recognition site and DNA end 5) Purify PCR product prior to restriction digest (NEB #T1030)