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 labeled 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 favor the annealed state and higher temperatures favor 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 catalog 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.
The T4 ligase relatives: t4 rna ligase 1 and what it joins
The phage supplies more than one ligase and they are not interchangeable. One joins single stranded ends and is what an adapter ligation in a small RNA library depends on; the other prefers a nicked double strand with an RNA strand, which is what makes it the enzyme for splinted ligations and for some structure probing methods. Both are inhibited by the things that inhibit the DNA enzyme and both are far more sensitive to the ATP concentration, so a reaction that works for DNA is a poor starting point. Read which substrate the supplier's unit definition used, because the same number means different work on each.
Where t4 pnk and t4 rna ligase 2 come into the same reaction
The kinase is the enzyme that makes an end ligatable at all: a fragment left by a nuclease or a synthetic oligonucleotide often carries no phosphate at its five prime end, and no ligase will join it. Adding the kinase before or with the ligation solves that, and it also removes a three prime phosphate where one is in the way. The reason to know this is that a ligation which fails for no visible reason is usually an end problem rather than an enzyme problem, and the test is cheap: treat the ends and repeat.
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.