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Home > Products > RNAi and RNA Enzymes > RNA Ligases > T4 RNA Ligase 2, truncated T4 RNA Ligase 2, truncated Catalog # Size Concentration Price Qty   M0242L 10,000 units 200,000 units/ml $240.00 M0242S 2,000 units 200,000 units/ml $60.00 Prices are in US dollars and valid only for US orders. Download: MSDS PDF Isolated from a recombinant source Supplied with 10X Reaction Buffer Requires 5' pre-adenylated RNA or DNA for ligation Description: T4 RNA Ligase 2, truncated (T4 RNL2 truncated) specifically ligates the pre-adenylated 5´ end of DNA or RNA to the 3´ end of RNA. The enzyme does not require ATP for ligation but does need the pre-adenylated substrate. T4 RNL2 truncated is expressed from a plasmid in E. coli which encodes the first 249 amino acids of the full length T4 RNA Ligase 2. Unlike the full length ligase, T4 RNL2 truncated is unable to adenylate the 5´ end of the substrate, and as a result it cannot ligate the phosphorylated 5´ end of RNA or DNA to the 3´ end of RNA (1-3). This enzyme, also known as RNL2 (1-249) has been used for optimized linker ligation for the cloning of microRNAs. This enzyme reduces background ligation because it can only use adenylated primers (4-5). Source: An E. coli strain that carries the cloned truncated T4 RNA Ligase 2 gene. Applications: Join a single stranded adenylated primer to small RNAs for cloning Selectively ligate an adenylated oligo in an RNA:DNA hybrid miRNA cloning Reagents Supplied: T4 RNL2 truncated Reaction Buffer (10X) Enzyme Properties Heat Inactivation: No Reaction & Storage Conditions Reaction Conditions: 1X T4 RNL2 truncated Reaction Buffer Incubate at 37°C. 1X T4 RNL2 truncated Reaction Buffer: 50 mM Tris-HCl 2 mM MgCl2 1 mM DTT pH 7.5 @ 25°C Unit Definition: One unit is defined as the amount of enzyme required to give 50% ligation of a 17-mer RNA to the pre-adenylated end of a 23-mer DNA when both 17- and 23-mer are annealed to a complementary 40-mer DNA strand in a total reaction volume of 20 µl in 30 minutes at 37°C. Unit Assay Conditions: 1X T4 RNL2 Truncated Reaction Buffer and 0.4 µg of an equimolar mix of the 23-mer, 17-mer  and 40-mer RNAs. After incubation at 37°C for 30 minutes, the ligated product is detected on a 20% denaturing polyacrylamide gel. Concentration: 200,000 units/ml Storage Conditions: 10 mM Tris-HCl 100 mM NaCl 0.1 mM DTT 0.1 mM EDTA 50% Glycerol pH 7.5 @ 25°C Storage Temperature: -20°C Notes Usage notes: For ligation of ssRNA to an adenylated DNA primer we recommend the following concentrations of reagents in a 20 µl reaction: 50-200 units of truncated T4 RNA Ligase 2 900 ng (150 picomoles) Universal miRNA Cloning Linker (NEB #S1315) 12% PEG 8000 MW 1X T4 RNL2 Truncated Reaction Buffer 200 ng ssRNA When incubated for 1-2 hours at room temperature, these conditions result in 70-100% ligation of 21-mer single-stranded RNA. The ligation product was measured on a denaturing 20% acrylamide gel. High concentrations of the adenylated DNA oligo are important for efficient ligation. Quality Control for Current Lot Quality control values for a specific lot can be found on the datacard which accompanies each product. Exonuclease Activity: Incubation of a 50 μl reaction containing 25 units of T4 RNA Ligase 2, truncated with 1 μg of a mixture of single and double-stranded [3H] E. coli DNA (205 cpm/μg) for 4 hours at 37ºC released < 0.1% of the total radioactivity. Endonuclease Activity: Incubation of a 50 μl reaction containing 25 units of T4 RNA Ligase 2, truncated with 1 μg of ΦX174 RF I DNA for 4 hours at 37ºC resulted in < 10% conversion to RFII as determined by agarose gel electrophoresis. RNase Assay: Incubation of a 50 µl reaction containing 25 units of T4 RNA Ligase 2, truncated with 3 µg of dsRNA ladder for 3 hours at 37°C resulted in no detectable degradation of the RNA as determined by agarose gel electrophoresis. Phosphatase Activity: Incubation of 25 units of Truncated T4 RNA Ligase 2 with 1 µg p-nitrophenyl phosphate (PNPP) in 50 µl T4 RNL2 truncated Reaction Buffer for 3 hours at 37°C released less than 0.05 µmol inorganic phosphate. References Ho, C.K. et al. (2004) Structure, 12, 327-339. Ho, C.K. and Shuman, S. (2002) Proc. Natl.Acad.Sci. USA, 99, 12709-12714. Nandakumar, J. et al. (2004) J. Biol. Chem, 279, 31337-31347. Aravin, A. and Tusch, T. (2005) FEBS Letters, 579, 5830-5840. Pfeffer, S. et al. (2005) Nat. Meth, 2, 269-276.