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T4 RNA Ligase

LO certified Recombinant enzyme Thermal inactivation at 70°C in 10 min

T4 RNA Ligase catalyzes the ATP-dependent formation of phosphodiester bonds between 5'-P and 3'-OH termini of oligonucleotides, single-stranded RNA and DNA.
  
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Thermo Scientific T4 RNA Ligase catalyzes the ATP-dependent intra- and intermolecular formation of phosphodiester bonds between 5'-phosphate and 3'-hydroxyl termini of oligonucleotides, single-stranded RNA and DNA.

The minimal substrate is a nucleoside 3',5'-biphosphate in intermolecular reaction and oligonucleotide of 8bases in intramolecular reaction.

Applications

  • RNA 3'-end labeling with cytidine 3',5'-bis [alpha-32P] phosphate (seeReference1)
  • Joining RNA to RNA(seeReference2)
  • Synthesis of oligoribonucleotides and oligodeoxyribonucleotides(seeReferences 3, 4)
  • Specific modifications of tRNAs(seeReference5)
  • Oligodeoxyribonucleotide ligation to single-stranded cDNAs for 5' RACE (Rapid Amplification of cDNA Ends)(seeReference6)
  • Site-specific generation of composite primers for PCR(seeReference7)

Note

The recommended BSA concentration in the reaction mixture is 0.1mg/mL.

  
Storage Condition-20 C
HazardousNo
10X Reaction Buffer500mM Tris-HCl (pH7.5 at 25°C), 100mM MgCl2, 100mM DTT, 10mM ATP
Concentration10 U/µL
Definition of Activity Unit
  • One unit of the enzyme catalyzes the conversion of 1nmol of 5'-[32P]-(A)12-18 to a phosphatase-resistant form in 30min at 37°C.
  • Enzyme activity is assayed in the following mixture: 50mM Tris-HCl (pH7.5), 10mM MgCl2, 10mM DTT, 1mM ATP, 10µM 5'-[32P]-(A)12-18 (10µM in 5'-termini).
InactivationInactivated by heating at 70°C for 10min.
InhibitionInhibitors: metal chelators, SH group-modifying reagents (8)
Molecular Weight43.6kDa monomer
Quality ControlThe absence of ribonucleases, exodeoxyribonucleases, endodeoxyribonucleases, and phosphatases confirmed by appropriate quality tests.
SourceE.coli cells with a cloned gene 63 of bacteriophage T4
Storage BufferThe enzyme is supplied in 20mM Tris-HCl (pH7.5), 1mM DTT, 50mM KCl, 0.1mM EDTA, 0.03%(v/v) ELUGENT Detergent and 50%(v/v) glycerol.

References

  1. O. C. Uhlenbeck, R. I. Gumport, T4 RNA ligase, The Enzymes (Boyer, P.D., Ed., Academic Press Inc., New York, 15B, 1982), pp. 31-60.
  2. T. Middleton et al., Synthesis and purification of oligoribonucleotides using T4 RNA ligase and reverse-phase chromatography.Anal. Biochem.144, 110-117 (1985).
  3. C. A. Brennan et al., Using T4 RNA ligase with DNA substrates.Meth. Enzymol.100, 38-52 (1983).
  4. D. C. Tessier et al., Ligation of single-stranded oligodeoxyribonucleotides by T4 RNA ligase.Anal. Biochem.158, 171-178 (1986).
  5. T. G. Heckler et al., T4 RNA ligase mediated preparation of novel "chemically misacylated'' tRNAPhes.Biochemistry23, 1468-1473 (1984).
  6. J. B. Edwards et al., Oligodeoxyribonucleotide ligation to single-stranded cDNAs: a new tool for cloning 5'-ends of mRNAs and for constructing cDNA libraries by in vitro amplification.Nucleic Acids Res.19, 5227-5232 (1991).
  7. S. Kaluz et al., Enzymatically produced composite primers: an application of T4 RNA ligase-coupled primers to PCR.BioTechniques.19, 182-186 (1995).
  8. H. M. Eun, Enzymology Primer for Recombinant DNA Technology (Academic Press. Inc., 1996).

MSDS & Certificates

pdf   T4 RNA Ligase MSDS
pdf   MSDS (JP) - T4 DNA Ligase

Protocols and Product Manuals

pdf   RNA 3'-end Labeling by Ligation Protocol
pdf   T4 RNA Ligase