Ligation of RNA molecules with T4 RNA ligase

Summary

T4RNA ligases have been used to generate many site-specific modified RNAs, especially oligonucleotide-modified and tRNA-modified with antisense codon tRNAs.In addition, T4RNA ligases have been used for intramolecular/intermolecular ligation of RNA/DNA, ligation of methylated oligodeoxyribonucleotides, cloning of full-length cDNAs, and incorporation of unnatural amino acids into protein molecules.

Operation method

Ligation of RNA molecules with T4 RNA ligase

Materials and Instruments

T4RNA Ligase Donor RNA Acceptor RNA
10XT4RNA ligase buffer Nuclease-free water FEG RNAase inhibitor

Move

I Materials and equipment

1) 10XT4RNA ligase buffer: 50 mmol/LTris (pH 7,8), l0 mmol/L MgCl₂, 5 mmol/LDTT,1 mmol/L ATP

2) Nuclease-free water

3) FEG (polyethyleneglycol) 40%

4)RNAase inhibitor

5)T4RNA ligase

6) Donor RNA

7) Acceptor RNA


(ii) Methods of operation

1) The reaction system for joining RNA to RNA molecules is as follows:

Donor RNA 100-500ng

acceptor RNA 250ng

10XT4RNA Ligase Buffer 4ul

RNasin Nuclease Inhibitor (40U/ul) 1ul

PEG, 40% 20ul

T4RNA Ligase (9-12U/u1) 1ul

Make up nuclease-free water to a final volume of 4 u1

2) Incubate at 37℃ for 30 min, or incubate at 16℃ overnight.

3) Recover by purification and store at 20 °C.

Caveat

1) Donor RNA molecules such as Poly(A)+RNA) must contain a 5' phosphate group.RNA) must contain a 5' phosphate group.2) The RNA molecule can be efficiently phosphorylated by poly(A) + RNA.

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