Enzymes for Molecular Biology: Enzymes for Molecular Biology

T4 RNA Ligase 2, Deletion Mutant, also known as T4Rnl2(1-249), is used to ligate single-stranded adenylated DNA or RNA oligonucleotides to small RNAs for cloning or next-generation RNA sequencing. The preadenylated 5´ ends of DNA or RNA are ligated to the 3´ ends of RNA. Unlike the full-length enzyme, T4Rnl2(1-249) is unable to adenylate the 5´ end of the substrate in the presence of ATP. However, it can use a preactivated donor (App-DNA or App-RNA) and join it to the 3´ end of an acceptor; thus, performing the ligation reaction in the absence of ATP which prevents circularization and other undesirable bimolecular reactions.

The enzyme is available in 2,000 and 10,000-Unit sizes at a concentration of 200 U/µl. The enzyme is supplied with a 10X Reaction Buffer.

Unit Definition: One unit is the amount of enzyme required to give 50% ligation of a 22-mer RNA to the preadenylated end of a 17-mer DNA when both oligos are annealed to a complementary 39-mer DNA strand in 30 minutes at 37°C under standard assay conditions.

Storage Buffer: 50% glycerol containing 50 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 0.1 mM EDTA, 1 mM dithiothreitol (DTT), and 0.1% Triton® X-100.

T4 RNA Ligase 2, Deletion Mutant, 10X Reaction Buffer: 500 mM Tris-HCl (pH 7.5), 20 mM MgCl₂, and 10 mM DTT.

Activity Assay: The unit definition assay is performed in a reaction containing 50 mM Tris-HCl (pH 7.5), 2 mM MgCl₂, 1 mM DTT and 0.4 µg of equimolar mix of 22-mer, 17-mer and 39-mer oligonucleotides, and varying amounts of enzyme.

Quality Control: T4 RNA Ligase 2, Deletion Mutant, is free of detectable DNA exo- and endonuclease, and RNase activities.