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:
Improper molar ratio of the vector DNA to the insert DNA.
Bad A-tailed insert DNA.
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:
Improper ligation reaction.
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:
Ampicillin is inactive.
IPTG/X-Gal is insufficient or inactive.
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