Product description: T4 RNA Ligase 2, also known as T4 Rnl2, has both intermolecular and intramolecular RNA strand joining activity. T4 RNA Ligase 2 is much more active joining nicks on double stranded RNA than on joining the ends of single stranded RNA. The enzyme requires an adjacent 5' phosphate and 3' OH for ligation. The enzyme can also ligate the 3' OH of RNA to the 5' phosphate of DNA in a double stranded structure.
Concentration and Size: 10U/μL
Unit Definition: One unit is defined as the amount of enzyme required to ligate 50% of 0.8μg of an equimolar mix of a 28-mer and 18-mer RNAs in a total reaction volume of 20μL in 30 minutes at 37℃.
Storage Buffer: 10mM Tris-HCl, 50mM KCl, 35mM (NH4)2SO4, 0.1mM DTT, 0.1mM EDTA, 50%(v/v) Glycerol, pH7.5@25℃.
T4 RNA ligase 1, T4 RNA ligase 2, RNaseR on enzymatic synthesis of circular RNA:
IVT reaction to obtain linear RNA: It is worth noting that for this step of IVT reaction, a certain proportion of GMP needs to be doped to obtain GMP-containing linear RNA;
RNA cyclisation: circular RNA is produced by joining the two ends within the RNA molecule by T4 RNA ligase 1 (T4 Rnl 1) or T4 RNA ligase 2 (T4 Rnl 2) catalysing the joining of 5'-phosphate and 3'-hydroxyl groups at the RNA terminus;
Removal of linear RNA: RNaseR is able to remove all linear RNA molecules from the reaction solution, resulting in linear RNA-free circular RNA.
Note: T4 RNA ligase2 requires ATP. (ATP, 100mM Solution, Cat#R1331)
Storage Conditions: Stored at -20±5℃. Do not store in a frost-free freezer. Always avoid freeze-thaw cycles or exposure to frequent temperaturechanges. These fluctuations can greatly alter product stability.
Quality Assurance:
1. Contaminant Assay: Free of Endonuclease, Exonuclease and RNase activities.
2. Endotoxin Assay: Pass.
3. Physical Purity: >95% by SDS-PAGE with Coomassie® blue staining.
