Double digest band pattern does not match prediction (Plasmid Construction (Double Digest))

Question

Accepted Answer

Picked colonies grow normally but the diagnostic double-digest yields bands of unexpected size on the gel. Common causes: 1) Restriction digest was incomplete (vector or insert not cut to completion) 2) Insert fragment size differs from expectation 3) PCR amplification produced non-specific products and the wrong fragment was cloned 4) An additional cryptic restriction site exists in the insert or vector 5) Wrong colony was picked, or the plate had mixed colonies Solutions: 1) Re-confirm digest conditions; extend incubation or add more enzyme 2) Re-amplify the insert and verify fragment size before ligation; gel-purify 3) Send the construct for full sequencing to confirm the design 4) Switch to a different restriction enzyme pair or redesign the cut sites 5) Re-pick single colonies on a fresh streak plate and re-screen