T4 DNA Ligase

T4 DNA ligase catalyzes the formation of phosphodiester bonds between adjacent 5'-phosphate and 3'-hydroxyl ends in double-stranded DNA or RNA. This enzyme can repair single-strand nicks in double-stranded DNA, RNA, or DNA/RNA hybrids. It can also join DNA fragments with sticky or blunt ends, but is inactive toward single-stranded nucleic acids. 
T4 DNA ligase requires ATP as a cofactor.

 

Application:

Cloning of restriction enzyme generated DNA fragments.

Cloning of PCR products.

Joining of double-stranded oligonucleotide linkers or adaptors to DNA.

Site-directed mutagenesis.

Amplified fragment length polymorphism (AFLP).

Ligase-mediated RNA detection.

Nick repair in duplex DNA, RNA or DNA/RNA hybrids.

Self-circularization of linear DNA

Reaction Conditions:               1×T4 DNA Ligase Reaction Buffer. Incubate at 16 °C.

 

Inhibition and Inactivation:   T4 DNA Ligase is strongly inhibited by NaCl or KCl at concentrations more than 200 mM.

                                                  Inactivated by heating at 65 °C for 10 min or at 70 °C for 5 min.

One Weiss unit of the enzyme catalyzes the conversion of 1 nmol of [32PPi] into Norit-adsorbable form in 20 min at 37°C. Enzyme activity is assayed in the following mixture: 66 mM Tris-HCl (pH 7.6), 6.6 mM MgCl2, 0.066 mM ATP, 10 mM DTT, 3.3 µM [32PPi].1 Weiss Unit = 200 CEUOne CEU is defined as the amount of enzyme required to give 50% ligation of HindIII fragments of lambda DNA in 30 min at 16°C.