a. The Quick Ligation reaction was heat inactivated. Heat inactivation in the presence of PEG, which is contained in the Quick Ligation buffer, inhibits transformation.
b. The Quick Ligation mix was not purified prior to electroporation. PEG in the Quick Ligation buffer prevents electroporation. Ligation products can be purified using a commercially available spin column.
c. The Quick Ligation was incubated overnight. PEG in the Quick Ligation buffer may cause a decrease in transformation efficiency over time. There is no addition benefit of letting the Quick Ligation reaction go beyond 30 minutes.
d. Ligation or transformation was inhibited by high salt in the reaction. Clean up the DNA.
e. Phosphatase (CIP , BAP or rSAP) was not completely inactivated or removed following dephosphorylation step. Follow the recommended procedure to remove the phosphatase or use Antarctic Phosphatase (NEB #M0289) or Shrimp Alkaline Phosphatase (rSAP) (NEB #M0371), which can be completely heat inactivated in 5 minutes at 70°C.
Other factors include:
i. Degraded ATP in buffer older than one year. Add new ATP to a concentration of 1 mM.
ii. Ligation produced only linear molecules because the DNA concentration was too high. We recommend a total DNA concentration of 1-10 µg/ml.
iii. The insert and the vector do not have phosphates.
iv. Too much ligation mixture was added to the cells. We recommend adding 1-5 µl to 50 µl of competent cells.
v. The insert was larger than 10 kb and the conditions didn?t allow circularization. Reduce the insert concentration.
vi. The restriction enzyme did not cleave efficiently. When cleaving near the end of a PCR fragment, we recommend leaving at least 6 base pairs on either side of the restriction site.
vii. The restriction enzyme was not completely inactivated. Phenol/ETOH purify the DNA if the enzyme can not be heat inactivated.
viii. Star activity from the restriction digest cleaved the vector or insert. Check the DNA on a gel. If there are extra bands, reduce the amount of enzyme or time for the restriction digest.