T4 RNA ligase.
Description
T4 RNA ligase (EC 6.5.1.3) forms a phosphodiester bond between a phosphorylated 5´ terminus and a 3´ hydroxyl of the ssRNA or ssDNA molecules. The minimum size of the 5´ moiety is a ribonucleoside 3´5´-bis phosphate, a 5´-monophosphate being ineffective; the minimum size of the 3´ moiety is a trinucleoside bis-phosphate.
Unit definition

1 Unit ligates 1 pmol of 32P-labelled mononucleoside biphosphate to a larger TCA-precipitable RNA species in 30 min at 4°C. Available at specific activities of 1000-4000 U/mg protein corresponding to 1-4 U/µl.

Reaction buffers
B15 (10x): 500 mM Tris-HCl 7.8, 100 mM MgCl2, 50 mM DTT, 10 mM ATP. Additional reagents: 50% PEG8000; 10 mM (NH4)3CoCl2.
Reaction conditions
50 mM TrisHCl 8.0, 10 mM MgCl2, 20 mM DTT, 1 mM ATP, 10% DMSO, 10-100 mg/ml BSA, 1-16 U RNA ligase, 0,5-20 mg RNA (1-100 mM substrate termini), 37íC for 30 min (or at 4íC overnight), terminated by boiling for 2 min.
Storage
Store at -20°C.
Applications
cDNA cloning; synthesizing oligomers; 3´ end-labeling of an RNA moiety; circularization of RNA and DNA oligonucleotides; joining RNA and ssDNA/dsDNA molecules.

Quality control

activity, SDS-PAGE purity, absence of RNase and DNase activities, specific performance tests.

References

1. England, T. et al. (1977) Proc. Natl. Acad. Sci. USA 74, 4839-4842. 2. Tessier, D.C. et al. (1986) Anal. Biochem. 158, 171-178. 3. Zhang, X.H., Chiang, V.L. (1996) Nucl. Acids Res. 24, 990-991. 4. Kinoshita, Y. et al. (1997) Nucl. Acids Res. 25, 3747-3748.

Catalog #
M3.4

 

 

Pack size

1.000 U

 

 

 

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