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