T4 RNA Ligase

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

Catalog#	Size, concentration	Supplied with:		Certificate of Analysis	MSDS
		10X Reaction Buffer	1 mg/ml BSA Solution
EL0021	1000 u (10 u/µl)	0.20 ml	0.20 ml	EL0021

Product information

Applications

RNA 3'-end labeling with cytidine 3',5'-bis [alpha-³²P] phosphate (1).
Joining RNA to RNA (2).
Synthesis of oligoribonucleotides and oligodeoxyribonucleotides (3, 4).
Specific modifications of tRNAs (5).
Oligodeoxyribonucleotide ligation to single-stranded cDNAs for 5' RACE (Rapid Amplification of cDNA Ends) (6).
Site-specific generation of composite primers for PCR (7).

Description

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 8 bases in intramolecular reaction.

Source

E.coli cells with a cloned gene 63 of bacteriophage T4.

Molecular Weight

43.6 kDa monomer.

Definition of Activity Unit

One unit of the enzyme catalyzes the conversion of 1 nmol of 5'-[³²P]-(A)_12-18 to a phosphatase-resistant form in 30 min at 37°C.

Enzyme activity is assayed in the following mixture: 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 10 mM DTT, 1 mM ATP, 10 µM 5'-[³²P]-(A)_12-18 (10 µM in 5'-termini).

Storage Buffer

The enzyme is supplied in:
20 mM Tris-HCl (pH 7.5), 1 mM DTT, 50 mM KCl, 0.1 mM EDTA, 0.03% (v/v) ELUGENT Detergent and 50% (v/v) glycerol.

10X Reaction Buffer

500 mM Tris-HCl (pH 7.5 at 25°C), 100 mM MgCl₂, 100 mM DTT, 10 mM ATP.

Quality Control

The absence of ribonucleases, exodeoxyribonucleases, endodeoxyribonucleases and phosphatases confirmed by appropriate quality tests.

Inhibition and Inactivation

Inhibitors: metal chelators, SH group-modifying reagents (8).
Inactivated by heating at 70°C for 10 min.

Note

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

Patents, Licenses, Trademarks

Protocols & recommendations

RECOMMENDATIONS FOR USE

ADDITIONAL PROTOCOLS

RNA 3'-end Labeling by Ligation

Prepare the following in a single RNase-free microfuge tube:

Total volume	20 µl
10X ligation buffer for T4 RNA Ligase	2 µl
10 mM ATP	1 µl
RNA	50-100 pmol
[³²P]-pCp	50-100 pmol (equimolar amount)
T4 RNA Ligase	1 µl (10 u)
DEPC-treated Water	to 20 µl

Incubate at 4°C for 10-12 hours (overnight).
Separate labeled RNA from unincorporated label by gel filtration on Sephadex G-50.

References

Uhlenbeck, O.C., Gumport, R.I., T4 RNA ligase, The Enzymes (Boyer, P.D., Ed.), Academic Press Inc., New York, 15B, 31-60, 1982.
Middleton, T., et al., Synthesis and purification of oligoribonucleotides using T4 RNA ligase and reverse-phase chromatography, Anal. Biochem., 144, 110-117, 1985.
Brennan, C.A., et al., Using T4 RNA ligase with DNA substrates, Meth. Enzymol., 100, 38-52, 1983.
Tessier, D.C., et al., Ligation of single-stranded oligodeoxyribonucleotides by T4 RNA ligase, Anal. Biochem., 158, 171-178, 1986.
Heckler, T.G., et al., T4 RNA ligase mediated preparation of novel “chemically misacylated’’ tRNAPhes, Biochemistry, 23, 1468-1473, 1984.
Edwards, J.B., 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.
Kaluz, S., et al., Enzymatically produced composite primers: an application of T4 RNA ligase-coupled primers to PCR, BioTechniques, 19, 182-186, 1995.
Eun, Hyone-Myong, Enzymology Primer for Recombinant DNA Technology, Academic Press. Inc., 1996.

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