T4 RNA Ligase 1 (ssRNA Ligase)

Description

Description:
T4 RNA Ligase 1 catalyzes the ligation of a 5′-phosphoryl-terminated nucleic acid donor to a 3′-hydroxyl-terminated nucleic acid acceptor through the formation of a 3′ -5′ phosphodiester bond, with hydrolysis of ATP to AMP and PP_i. Substrates include single-stranded RNA and DNA as well as dinucleoside pyrophosphates.

Applications:

• Labeling of 3′-termini of RNA with 5′-[³²P] pCp
• Inter- and intramolecular joining of RNA and DNA molecules
• Synthesis of single-stranded oligodeoxyribo-nucleotides
• Incorporation of unnatural amino acids into proteins
• Ligation of ssRNA and DNA

During ligation of single-stranded DNA to the 3′ end of RNA, a 5′-phosphorylated 3′-amino-blocked 30-mer DNA oligonucleotide was incubated with 5′ FAM-labeled 15-mer RNA at 25 °C for 4 hours. Under these conditions, 20 units of MCLAB T4 RNA Ligase 1 efficiently ligated the 30-mer ssDNA to the 3′ end of the RNA substrate.

Top graph: Substrate 5′ FAM-labeled 15-mer RNA (blue trace).
Bottom graph: Incubation with T4 RNA Ligase 1 yields a 45-nt RNA–ssDNA product.
Orange peaks: DNA Size Standard

Source:
An E. coli strain that carries the T4 RNA Ligase 1 gene

1x T4 RNA Ligase Reaction Buffer (and 10mM ATP):
50 mM Tris-HCl
10 mM MgCl₂
1 mM DTT
pH 7.5 @ 25°C

Heat Inactivation:
65°C for 15 minutes
Heat inactivation by boiling for 2 minutes

Reaction Conditions:
1x T4 RNA Ligase Reaction Buffer
Supplemented with 1 mM ATP
Incubate at 37°C

Unit Definition:
One unit is defined as the amount of enzyme required to convert 1 nmol of 5´-[³²P]rA16 into a phosphatase-resistant form in 30 minutes at 37°C.

Unit Assay Conditions:
1x T4 RNA Ligase reaction buffer, supplemented with 1 mM ATP, is mixed with the RNA substrate (10µM of 5′-[³²P]rA16 ) and varying amounts of enzyme. Incubation is at 37°C for 15 minutes.

Concentration: 20,000 units/ml

Specific Activity: 168,000 U/mg

Storage Conditions:
10 mM Tris-HCl
50 mM KCl
1 mM DTT
0.1 mM EDTA
50% Glycerol
pH 7.5 @ 25°C

Recommended Storage Condition: -20°C

Additional information

Manual & Protocols

The Q&A for this product will be available soon.