Cloning: empty vector

1. Ranked causes

   1. Insert DNA degraded or too dilute for ligationInsert band weak/absent on gel; fresh PCR/gel-extract restores recombinants.
   2. Incomplete vector digest / uncut plasmid backgroundNo-insert ligation plate also crowded; vector looks mostly circular on gel.
   3. Insert:vector molar ratio far from optimalAdjusting to ~3:1 insert:vector raises recombinant fraction.
   4. Phosphatase omitted on sticky-end vector (re-ligation favoured)CIP/SAP treatment of vector drops empty background sharply.

2. Recovery steps

   1. Re-digest vector longer; gel-purify linear band; phosphatase-treat sticky endsCuts the uncut/re-ligation background that floods empty clones.
   2. Re-prep insert; confirm concentration and size before ligationInvisible/degraded insert presents as all-empty screens.
   3. Set insert:vector ≈ 3:1 molar and include a no-insert control plateRatio and control plate quantify how much empty background to expect.

3. Controls first

   • Did the no-insert ligation control plate also yield many colonies?If yes: Vector self-ligation / incomplete cut — treat vector prep before blaming insert.

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