Ligations are very inefficient and easily inhibited by impurities such as agarose, other enzymes, excess DNA etc. Therefore pay attention to DNA purity and concentrations.
ATP goes off!! Do not store it diluted and do not freeze and thaw buffer too many times. If you use the commercial buffer that has ATP in it aliquot it or keep track of how many times you thaw it.
Blunt end ligations are inhibited by ATP
Kinase buffer and Ligase Buffer are almost the same and are compatible
Always include the following controls :
1) Ligation control = single cut vector alone. This should ligate very efficiently.
2) Control SAPing and degree of background due to uncut vector = set up ligations containing each cut and sapped vector with and without insert.
Ligate at 16oC o/n in small eppendorfs in the PCR machine to avoid evaporation.
Insert3X molar ratio to vector
H2O to final vol 20l