T4 DNA ligase joins DNA ends, and it is the enzyme behind most cloning that is not done by assembly chemistry. Buying it looks trivial until two suppliers' units turn out to mean different things and a reaction that worked for years stops working after a buffer change. This page is about the unit definitions, the buffer, the conditions that actually matter, and what separates a cheap tube from an expensive one.
Two unit definitions, and why they are not comparable
Suppliers define ligase activity in more than one way, classically by the conversion of a labelled substrate under stated conditions, and the resulting figures differ by a large factor between definitions. A protocol that calls for a number of units is therefore only meaningful alongside the supplier it was written against. When changing supplier, titrate rather than converting: run the same ligation at several enzyme amounts and pick the one that works, and write down which product and which lot it was.
The buffer is half the reaction
Ligase needs adenosine triphosphate and magnesium, and the supplied buffer provides both. That buffer is also the part that degrades: repeated freeze and thaw cycles hydrolyse the nucleotide, which is the commonest cause of a ligation that used to work and now does not. Keep single-use aliquots, and be suspicious of the buffer before the enzyme. Some crowding-agent formulations shorten incubations dramatically and change the optimum enzyme amount, so they are a different reaction rather than a faster one.
Temperature, time and the ends being joined
Blunt ends, single-base overhangs and long cohesive overhangs are three different problems. Long overhangs anneal and ligate readily; blunt ligation is slow and benefits from more enzyme and longer incubation; a single-base overhang sits between the two. Lower temperatures favour the annealed state and higher temperatures favour enzyme turnover, which is why a long incubation in the cold and a short one at room temperature can both be right. Match the condition to the ends, and do not carry one laboratory protocol across all three.
What separates the products, and when it matters
Concentration, whether the enzyme is supplied with a crowding buffer, and whether a compliant manufacturing grade exists in the same line are the real differences; the catalogue price per unit is the least informative number. High concentration matters when the reaction volume is small or the ends are difficult. A documented grade matters only when the product of the reaction is heading somewhere regulated, in which case the enzyme's documentation becomes part of the record rather than a convenience.
Questions people ask about t4 dna ligase
What is a unit of T4 DNA ligase?
An activity defined by the supplier's own assay, and the common definitions differ substantially. Do not convert between suppliers: titrate the enzyme in your own reaction and record the product and lot.
Why did my ligation stop working?
Most often the buffer rather than the enzyme: the nucleotide in it degrades with repeated freeze and thaw. Use single-use aliquots of buffer before blaming the ligase.
Do I need the high concentration product?
When the reaction volume is small, the ends are blunt, or the insert is at low molar concentration. For routine cohesive-end cloning the standard concentration is sufficient.