Confirmed colony later shows recombination or deletion (Plasmid Construction)

Question

Accepted Answer

Initial screening looked correct, but on re-verification the insert is missing pieces or the vector has rearranged. Common causes: 1) Insert contains repeat sequences, LTRs, or other unstable elements that recombine 2) Insert is too long (push the host's tolerance) 3) Host strain is not suitable for the construct (recA+ standard strains recombine repeats) 4) Culture period was too long, increasing recombination probability Solutions: 1) Use a recombination-deficient strain such as Stbl3 / NEB Stable / SURE for repeats and viral backbones 2) Shorten culture time; grow at 30 °C instead of 37 °C 3) If possible, shorten the insert or redesign the cloning strategy 4) Sequence-verify critical constructs early and immediately make a glycerol stock