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Cat. No.: T4RL1-1k (for 1KU)

Cat. No.: T4RL1-5k (for 5KU)

Description

T4 RNA Ligase 1 is an ATP-dependent enzyme that catalyzes the formation of a phosphodiester bond between the 5'-P and 3'-OH ends of single-stranded RNA, single-stranded DNA, or nucleotide molecules. T4 RNA Ligase 1 exhibits the highest efficiency in connecting RNA molecules, followed by DNA-RNA connections, and the lowest efficiency in DNA-DNA connections. Therefore, it is also considered an ssRNA ligase.

T4 RNA Ligase 1 is primarily used for connecting RNA molecules, requiring the presence of a 5' phosphate group and a 3' hydroxyl group.

T4 RNA Ligase 1 can also be used for connecting RNA and nucleotides. The nucleotide must be phosphorylated at both the 5' and 3' ends, and this application is commonly used for labeling the 3' end of RNA.

T4 RNA Ligase 1 can also be used for connecting DNA and RNA. When DNA provides a 5' phosphate group and RNA provides a 3' hydroxyl group, the connection efficiency is relatively high. However, when DNA provides a 3' hydroxyl group and RNA provides a 5' phosphate group, the connection efficiency is very low.

T4 RNA Ligase can also be used for connecting DNA and DNA, but the efficiency is very low. It is mainly used for DNA circularization, such as in cDNA circularization in 5' RACE. Although DNA-DNA connections are possible, they are more challenging.

The catalytic process of T4 RNA Ligase 1 for the ligation reaction is as follows: First, T4 RNA Ligase 1 reacts with ATP, generating an intermediate product called T4 RNA Ligase 1-AMP and releasing pyrophosphate. Next, AMP is transferred from the intermediate product T4 RNA Ligase 1-AMP to the 5' phosphate end of the nucleic acid, forming an adenylated nucleic acid intermediate. Finally, under the catalysis of T4 RNA Ligase 1, the 3' hydroxyl group of another nucleic acid attacks the 5' phosphate end of the adenylated nucleic acid intermediate, forming a 3'-5' phosphodiester bond and releasing AMP.

Application

• Inter-molecular ligation of ssRNA
• Inter-molecular ligation of ssRNA and ssDNA
• Can be used for inter-molecular ligation of ssDNA, but with lower efficiency
• Labeling of ssRNA 3'-OH end with radioactive isotopes such as 5'-[32P]pCp
• Synthesis of single-stranded Oligo RNA and Oligo DNA
• Special modifications of tRNA
• Used in 5'-RACE (Rapid Amplification of cDNA Ends) experiments for oligonucleotide ligation to single-stranded cDNA
• Introduction of non-natural amino acids into proteins, etc.

Source

Recombinant protein expressed in Escherichia coli, with the gene derived from T4 bacteriophage. The enzyme has a molecular weight of approximately 44 kDa.

Activity definition

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

Purity

Free of DNA endonucleases, exonucleases, RNAse, and phosphatases.

Enzyme storage solution

10 mM Tris (pH 7.5), 200 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 50% (v/v) glycerol.

10X Reaction Buffer

500 mM Tris-HCl (pH 8.0 at 25°C), 100 mM MgCl2, 20 mM DTT.

Inactivation or inhibition

Heating at 65°C for 15 minutes or boiling for 2 minutes can inactivate T4 RNA Ligase 1. Addition of 0.5 M EDTA pH 8.0 at a volume of 10% can also terminate the reaction.

Precautions

• For the substrates to be ligated using this product, whether single-stranded RNA or single-stranded DNA, it is necessary to ensure that the 5' end is phosphorylated or adenylated, and the 3' end is hydroxylated.
• If the substrates to be ligated are double-stranded RNA or DNA, it is recommended to use products from other series such as T4 RNA Ligase 2.
• Depending on the specific application, appropriate operating methods can be selected, and additional reagents may need to be prepared, such as RNase inhibitor, DEPC water, etc.
• Depending on the type of ligation reaction, it is recommended to add an appropriate amount of PEG8000 to the reaction system. For ligation reactions between single-stranded RNA or DNA, the final concentration of PEG8000 in the reaction system is recommended to be 15%-25%. This significantly improves enzyme activity without affecting the reaction characteristics.
• This product is intended for scientific research by professionals only and should not be used for clinical diagnosis or treatment, food or drug purposes, or stored in residential settings.
• For your safety and health, please wear laboratory attire and disposable gloves when handling.

Storage

The minimum shelf life is 1 year at -20°C.

Related:

• T4 RNA Ligase
• T4 RNA Ligase 1 (ssRNA Ligase)
• T4 RNA Ligase 2 (dsRNA Ligase)
• T4 RNA Ligase 2, truncated
• T4 RNA Ligase 2, truncated KQ

SBS Genetech is recognized as one of the global major leading industry players in Ligase Market by third-party market researchers. For more details, please visit Ligase Market - A Global and Regional Analysis Focus on Product, Source, Application, End User, and Country - Analysis and Forecast, 2022-2032