T4 RNA Ligase catalyzes the ATP-dependent ligation of single-stranded RNA or DNA onto the 5’-phosphoryl termini of single-stranded RNA or DNA. Substrates include single-stranded RNA and DNA as well as dinucleoside pyrophosphates. The enzyme has a molecular weight of 43.5 kDa.

Source

Purified from E. coli strain containing the plasmid with T4 RNA ligase gene.

Applications

• Labeling of 3´ – termini of RNA with 5´ – [ ³² P] pCp
• Inter- and intramolecular joining of RNA and DNA molecules
• Synthesis of single-stranded oligodeoxyribonucleotides
• Incorporation of unnatural amino acids into proteins

Reaction Conditions

• 50 mM Tris-HCl (pH 7.8)
• 10 mM MgCl ₂
• 10 mM DTT
• 1 mM ATP

Incubate at 37°C.

Unit Definition

One unit is defined as the amount of enzyme required to convert 1 pmol of AMP to an acid-insoluble form in 10 minutes at 25°C.

Concentration

10-100,000 units/ml

Heat inactivation

65°C for 15 minutes or boiling for 2 minutes.

Quality Assurance

Each lot of T4 DNA Ligase is tested for endonucleases/exonucleases, in a blue/white cloning assay.

Storage buffer

• 50 mM KCl
• 10 mM Tris-HCl (pH 7.4)
• 0.1 mM EDTA
• 1 mM DTT
• 50% glycerol

Storage conditions

-20°C