T4 RNA Ligase
Cat. No. RP701
Store at -20°C
Size 1000 Units
- T4 RNA Ligase
- 10 X T4 RNA Ligase Buffer
Concentration 30 units/µL
T4 RNA Ligase catalyzes the ATPdependent formation of phosphodiester bonds between a donor with 5’-phosphonyl-terminated nucleic acid and an acceptor with 3’-hydroxyl-terminated nucleic acid1). The substrates include RNA, DNA, oligoribonucleotides, and oligodeoxyribonucleotides.
20 mM Tris-HCl (pH7.5)
50 mM NaCl
1 mM DTT
0.1 mM EDTA
50 % Glycerol
10 X T4 RNA Ligase buffer
550 mM HEPES-NaOH (pH7.5)
150 mM MgCl2
33 mM DTT
10 mM ATP
Recombinant protein, expressed in E.coli.
Recombinant T4 RNA
Ligase which has the C-terminal hexahistidine tag was expressed in E.coli, and purified by metal chelatingcolumn.
- 3’-End labeling of RNA 2)
- Ligation of RNA to RNA 3,4)
- Specific modification of tRNAs for incorporation of unnatural amino acids into proteins 5,6)
ProteinExpress determined the catalytic unit using aminoacylated pdCpA and tRNA lacking the 3’-terminal dinucleotide. One unit catalyzes 60% ligation of TAMRA-X-AF-pdCpA(40 pmol) with tRNAPhe(-CA) (14 pmol) at 4 °C for 2hr, which is equivalent to the conversion of 1 pmol of pCp into its acid-insoluble form in 10 minutes at 5 °C with oligo(A)n as the substrate.
A) Reagents to be supplied by user
- Nuclease-Free Water
- 0.1 % BSA
B) Ligation of single-stranded RNA
1. Prepare the following reaction mixture in a sterile microcentrifuge tube.
Single-stranded RNA (Donor) 100-500 ng
Single-stranded RNA (Acceptor) 250 ng
10 X T4 RNA Ligase buffer 5 µL
0.1 % BSA 1 µL
T4 RNA Ligase (30 units/µL) 1 µL
Nuclease-Free Water up to 50 µL
2. Incubate at 4-16 °C for 2-16 hr
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- Romaniuk, P.J. and Uhleback, O.C., Methods Enzymol.
- Middleton, T. et al., Anal Biochem., 144, 110 (1985) (1991).
- Robertson, S.A. et al., J. Am. Chem. Soc., 113, 2722
- Hohsaka, T. et al., J. Am. Chem. Soc., 121, 34 (1999).