T4 DNA Ligase Buffer

dATP is a competitive inhibitor. Phosphate will reduce ligation efficiency. Detergents in your ligation buffer will likely not affect activity. High levels (0.2M) Na2+, K+, Cs+, Li+, and NH4+ inhibit the enzyme almost completely. Polyamines, spermine, and spermidine also serve as inhibitors.

At least one molecule in a ligase reaction (i.e., insert or vector) must be phosphorylated. Ligation reactions are dependent on the presence of a 5' phosphate on the DNA molecules. The ligation of a dephosphorylated vector with an insert generated from a restriction enzyme digest (phosphorylated) is most routinely performed. Although only one strand of the DNA ligates at a junction point, the molecule can form a stable circle, providing that the insert is large enough for hybridization to maintain the molecule in a circular form.

For cloning an insert with one cohesive end and one blunt end, use the conditions for blunt ends.  The sticky end may ligate quickly, but the blunt end ligation will still be inefficient. You should use the more stringent protocol to optimize the blunt end ligation. This usually means using more enzyme (5 U), a lower reaction temperature (14C) and a longer incubation time (16-24 hours). 

Components of the ligation reaction (enzymes, salts) can interfere with transformation, and may reduce the number of recombinant colonies or plaques. We recommend a five-fold dilution of the ligation mix, and adding not more than 1/10 of the diluted volume to the cells. For best results, the volume added should also not exceed 10% of the volume of the competent cells that you are using.

Generally, ligations are done in a 20 µL volume. Use a total of 100 to 1000 ng of DNA with an insert to vector ratio of 3:1. Add 1.0 units (Weiss) ligase to the reaction. Incubate at room temperature for 4 h or overnight at 14-16 degrees C.

Ideally, assemble several reactions with varying ratios of vector:insert (i.e. 3:1, 5:1, 10:1, 20:1, etc.) to determine the optimal ratio for ligation.

Thermo Fisher Scientific offers T4 DNA ligase at two concentrations: 1 U/µL (Cat. No. 15224-017) and 5 U/µL (Cat. No. 15224-041). When performing blunt or TA cloning ligations, the higher concentration of ligase is generally preferred since ligating a blunt or single base overhang requires more enzyme.