Cloning: wrong insert

1. Ranked causes

   1. Primer mix-up or oligo order error (wrong amplicon)Sequencing matches a different target; re-ordered primers fix it.
   2. Wrong PCR product / gel band excised for the insertColony PCR size matches a neighbouring band from the gel extract.
   3. Partial digest leaving alternative fragments that ligateMultiple insert sizes across clones; complete digest collapses to one size.
   4. Annotation / map mismatch (expected size calculated wrong)Sequence is correct for the intended construct; map feature was misread.
   5. Cross-contamination from another ligation on the benchSequence matches a concurrent cloning project; fresh tips/tubes stop it.

2. Recovery steps

   1. Re-amplify insert; verify size on gel against ladder before ligationStops wrong-band excision from propagating into clones.
   2. Confirm oligo IDs/sequences against the design file; re-order if mismatchedOligo mix-ups look like successful cloning of the wrong thing.
   3. Sequence 2–3 colonies across the junctions before scaling miniprepsCatch wrong inserts before investing in midiprep/expression.

3. Controls first

   • Does colony-PCR / digest size match the positive-control insert size you intended?If no: Wrong band, primers, or partial digest — re-check insert prep before more screening.

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