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Cat. No.: T4RL2K-5k (for 5KU)

Cat. No.: T4RL2K-20k (for 20KU)

Cat. No.: T4RL2K-100k (for 100KU)

Description

T4 RNA Ligase 2, truncated KQ (T4 Rnl2tr KQ or T4 Rnl2tr R55K, K227Q) is a double-point mutant of T4 RNA Ligase 2, truncated, with the mutations R55K and K227Q. It greatly reduces the non-specific ligation of RNA by T4 RNA Ligase 2, truncated (such as RNA concatenation or self-circularization) while maintaining similar enzymatic activity to T4 RNA Ligase 2, truncated.

T4 RNA Ligase 2, truncated KQ is derived from T4 RNA Ligase 2, truncated (R0635) with mutations of glutamine at position 227 and lysine at position 55, replacing the original arginine and lysine, respectively. The K227Q mutation significantly reduces the activity of T4 RNA Ligase 2, truncated in transferring the adenylate group from the 5'-adenylated DNA adapter (AppDNA) to the RNA 5'-phosphate, forming a 5'-phosphorylated DNA adapter (pDNA). This reduction decreases the non-specific ligation of RNA caused by T4 RNA Ligase 2, truncated (such as RNA concatenation or self-circularization). However, the ligation activity of T4 RNA Ligase 2, truncated K227Q is significantly decreased. By further mutating R55K on T4 RNA Ligase 2, truncated K227Q, the ligation activity of the enzyme is restored to a level similar to that of the wild-type T4 RNA Ligase 2, truncated.

T4 RNA Ligase 2, truncated KQ exhibits similar effects as T4 RNA Ligase 2, truncated in ligating ssRNA and AppDNA. However, T4 RNA Ligase 2, truncated can deadenylate AppDNA to form pDNA, whereas T4 RNA Ligase 2, truncated KQ does not possess deadenylation activity on AppDNA.

T4 RNA Ligase 2, truncated KQ is commonly used for cloning, high-throughput sequencing library preparation, and PCR detection of single-stranded RNAs with a 3'-hydroxyl end, where a 5'-adenylated DNA or RNA adapter is added to the 3'-hydroxyl end.

T4 RNA Ligase 2, truncated KQ can only utilize 5'-adenylated single-stranded DNA or RNA as the 3'-end adapter, thus greatly reducing the background in the ligation reaction. It is often the preferred choice for small RNA library preparation.

T4 RNA Ligase 2, truncated KQ does not require ATP for ligation but relies on the 5'-adenylated substrate. Even in the presence of ATP, T4 RNA Ligase 2, truncated KQ cannot catalyze the formation of a phosphodiester bond between the 5'-phosphate end of single-stranded DNA or RNA and the 3'-OH end. T4 RNA Ligase 1, on the other hand, can catalyze this ligation in the presence of ATP. Therefore, T4 RNA Ligase 2, truncated KQ is particularly suitable for small RNA library preparation, whether it is for miRNA sequencing or directional mRNA-Seq. It improves the quality of library construction and effectively reduces the background in the ligation reaction.

Application

• Connecting 5'-pre-adenylated single-stranded DNA or RNA with the 3'-hydroxyl end of single-stranded RNA.
• In cDNA library construction, connecting 5'-pre-adenylated single-stranded oligonucleotides and small RNAs.
• In strand-specific cDNA library construction, connecting 5'-pre-adenylated single-stranded oligonucleotides and RNA.
• In second-generation sequencing, connecting the 3' end of RNA with adapters in miRNA library construction.
• In cDNA library construction for small RNA transcriptome analysis.

Source

Recombinant protein expressed in Escherichia coli, comprising the N-terminal amino acids 1-249 of T4 bacteriophage T4 RNA ligase 2 with R55K and K227Q double mutations.

Unit of activity

200 units is defined as the amount of enzyme required to achieve 80% ligation of a 31-mer RNA to the pre-adenylated end of a 17-mer DNA (Universal miRNA Cloning Linker) in a total reaction volume of 20 µl within 1 hour at 25°C.

Purity

No DNA endonuclease or exonuclease activity, no RNAase activity, no protease activity.

Enzyme storage solution

10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 0.1 mM DTT, 50% (v/v) glycerol.

Inactivation or inhibition

T4 RNA Ligase 2, truncated KQ can be inactivated by heating at 65°C for 20 minutes, or its activity can be inhibited by the addition of proteinase K or EDTA.

Precautions

• The reagents provided in this product are prepared with water that does not contain DNAase and RNAase and can be directly used for the ligation reaction between 3'-hydroxyl single-stranded RNA and 5'-adenylated DNA or RNA.
• Depending on the specific application, suitable operating methods can be selected, and additional reagents may be required, such as RNase Inhibitor and DEPC water.
• When using this product to ligate ssRNA and AppDNA, it is recommended to use a concentration of 10%-30% PEG8000. Increasing the concentration of PEG8000 appropriately will significantly improve the catalytic ligation efficiency of T4 RNA Ligase 2, truncated KQ without altering its reaction characteristics.
• This product is intended for scientific research purposes by professionals only and should not be used for clinical diagnosis or treatment, food or drug purposes, or stored in ordinary residences.
• For your safety and health, please wear lab coats 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