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Product description: T4 RNA Ligase 1 is an ATP-dependent ligase, active on a broad range of substrates including single-strand RNA, DNA, oligoribonucleotides, oligodeoxynucleotides, as well as numerous nucleotide derivatives. The enzyme catalyzes the formation of a phosphodiester bond between a 5'-phosphoryl-terminated nucleic acid donor to a 3'-hydroxyl-terminated nucleic acid acceptor in a template-independent manner.
Source: Recombinant E.coli
Concentration and Size: 10U/μL
Unit Definition: One unit is defined as the amount of enzyme required to ligate 50% of 0.4 ug of an equimolar mix of two single stranded 23 base RNA oligonucleotides (one 5'-phosphorylated) in 20 μL 1X T4 RNA Ligase Buffer following a 30 minute incubation at 37℃.
Storage Buffer: 10mM Tris-HCl (pH 7.5, 25℃), 0.1mM EDTA, 500mM KCl, 1mM DTT, and 50% (v/v)Glycerol.
Companion Product: 10X T4 RNA Ligase Buffer, Cat#ON-111, 500mM Tris-HCl pH7.8, 25℃, 100mM MgCl2, 10mM ATP, 100mM DTT.50% PEG 8000, Cat#ON-112, 50% PEG 8000(w/v).
Protocol for ligation of single-stranded RNA
1. Assemble the following reaction in a sterile microcentrifuge tube:
|10X T4 RNA Ligase Buffer||2μL|
|Ribonuclease inhibitor (40U/μl)||0.5μL|
|PEG 8000, 50%||8μL|
|T4 RNA ligase 1(10U/μL)||1μL|
|Nuclease-free H2O||to 20μL|
Note: Donor molecule must contain a 5'-phosphate group; Acceptor RNA must contain a 3'-OH group
2. Incubate the reaction at 37℃ for 30min or 16℃ overnight.
3. Stop reaction by adding EDTA to a final concentration of 11mM.
Storage Conditions: -20℃
Quality Assurance: Free of endonuclease, exonuclease and RNase activities.
Physical Purity: ＞95% by SDS-PAGE.
1. Thomas J. Snopek, William B. Wood, M. Patricia Conley, et al. Proc. Natl. Acad. Sci. USA. 74, 3355–3359 (1997).
2. Robert Silber, V.G. Malathi, and Jerard Hurwitz. Proc. Nat. Acad. Sci. USA. 69, 3009-3013(1972).
3. Uhlenbeck, O.C. and Gumport, R.I. (1982) In: The Enzymes, Boyer, P.D., ed., Academic Press, New York, NY.
4. Romaniuk, P.J. and Uhlenbeck, O.C. (1983) Joining of RNA molecules with RNA ligase. Methods Enzymol. 100, 52–9.