T4 RNA Ligase

Supplied Reagents

• T4 RNA Ligase
• 10 X T4 RNA Ligase Buffer

Description

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.
 

Image	Cat.No:	Size	Concentration	Storage	Link
	FNK-RP701	1000 Units	30 units/µL	-20°C
	FNK-RP702	5000 Units

Storage Buffer

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

Source

Recombinant protein, expressed in E.coli.

Additional Information

Recombinant T4 RNA
Ligase which has the C-terminal hexahistidine tag was expressed in E.coli, and purified by metal chelatingcolumn.

Applications

• 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)

Unit definition 

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. 

Standard Application

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

Reference

1. England, T.E. et al., Proc. Natl. Acad. Sci. USA, 74, 4839 (1977). Vol.15, Academic Press, New York, 31 (1982).
2. Uhlebeck, O.C. and Gumport, R.I., in The Enzymes, 100, 52 (1983).
3. Romaniuk, P.J. and Uhleback, O.C., Methods Enzymol. 
4. Middleton, T. et al., Anal Biochem., 144, 110 (1985) (1991).
5. Robertson, S.A. et al., J. Am. Chem. Soc., 113, 2722 
6. Hohsaka, T. et al., J. Am. Chem. Soc., 121, 34 (1999).