T&ATM Cloning
No colonies
Problems in transformation or use low-efficiency competent cells.
Less than 100 transformed colonies grow on selection plate. When checking the colonies with colony PCR using M13F&R primers, false inserts of 0.15 and 1.8 kb fragments are obtained.
  1. Improper molar ratio of the vector DNA to the insert DNA.
  2. Bad A-tailed insert DNA.
  3. The 0.15kb fragment is derived from reaction of self-ligation of the vector itself. The 1.8 kb fragment is derived from the residue in production process.
High colony number, but high percentages of blue colonies with insert DNA.
  1. Improper ligation reaction.
  2. DNA is inserted, but it’s not disrupting the expression of  LacZ gene or the insertion generates in-frame fusion of the coding region to lacZ gene.
No blue colonies are present on the plate.
  1. Ampicillin is inactive.
  2. IPTG/X-Gal is insufficient or inactive.
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