DNA ligation is the molecular biology technique which joins together the two ends of DNA molecules. This could be from the same DNA molecule as in the circularization of a plasmid, or the joining of two different DNA molecules, hence the term DNA cloning (the creation of new DNA molecules).
How Does DNA Ligation Work?
DNA ligation involves the creation of a phosphodiester bond bond between the 3' hydroxyl of one nucleotide and the 5' phosphate of another. The DNA ligation reaction could not proceed rapidly on its own and therefore it is catalyzed by the addition of a DNA ligase enzyme such as T4 DNA Ligase.
T4 DNA ligase enzyme catalyzes the ligatation of DNA fragments having blunt or overhanging, complementary, 'sticky' ends.
Typically, it is easier to ligate molecules with complementary sticky ends than blunt ends. T4 DNA ligase is the most commonly used DNA ligase for molecular biology techniques and can ligate 'sticky' or blunt ends.
The two components of the DNA in the ligation reaction should be equimolar and around 100μg/ml. Most commonly, one wants to ligate an insert DNA molecule into a plasmid, ready for bacterial transformation. Typically, DNA and plasmid vector are individually cut to yield complementary ends, then both are added to a ligation reaction to be circularised by DNA ligase. If the plasmid backbone to insert DNA ratio is too high then excess 'empty' mono and polymeric plasmids will be generated. If the ratio is too low then the result may be an excess of linear and circular homo- and heteropolymers.
DNA Ligation Protocol
For a 20μL Ligation Mix:
Larger ligation mixes are also commonly used:
1.0 μL 10X T4 ligase buffer
6:1 molar ratio of insert to vector (~10ng vector)
