Ligation

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.

 

Vector 50ng

Insert 3X molar ratio to vector

10XLigase Buffer 2l

10mm ATP 2l

T4 Ligase 1l

H2O to final vol 20l