T&ATM Cloning
Q1:
No colonies
A1:
Problems in transformation or use low-efficiency competent cells.
Q2:
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.
A2:
  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.
Q3:
High colony number, but high percentages of blue colonies with insert DNA.
A3:
  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.
Q4:
No blue colonies are present on the plate.
A4:
  1. Ampicillin is inactive.
  2. IPTG/X-Gal is insufficient or inactive.
 
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