T4 RNA Ligase 2, truncated KQ (C007B-566412)

Aladdin's T4 RNA Ligase 2, truncated KQ is a double mutant of truncated T4 RNA Ligase 2, but maintains similar enzyme activities to truncated T4 RNA Ligase 2. This product greatly reduces non-specific RNA ligation to prevent the formation of tandem repeats or RNA loop.This product is developed on the basis of truncated T4 RNA Ligase 2 , with R55K and K227Q mutation. The K227Q mutation greatly reduces the non-specific RNA ligation, but with a significant decrease in ligase activity. However with one more mutation of R55K, the ligase activity of this product is recovered and has similar ligation efficiency to the truncated T4 RNA Ligase 2.This product and T4 RNA Ligase 2, truncated have similar performance in ligating AppDNA to ssRNA, but T4 RNA Ligase 2, truncated can deadenylate AppDNA into pDNA, whereas this product does not have deadenylation activity on AppDNA.This product is commonly used to ligate the 5'-adenylated DNA or RNA adaptors to the 3'-OH end of single-stranded RNA, such as microRNAs (miRNAs), for cloning, library preparation for NGS sequencing, or PCR assays.This product requires the 5'-adenylated single-stranded DNA or RNA for ligation, thus greatly reducing the background of ligation reaction, and is often preferred for preparing small RNA libraries.The ligation reaction catalyzed by this product does not require ATP, but requires a 5'-adenylated substrate. Even in the presence of ATP, this product cannot catalyze the formation of phosphodiester bonds between the 5'-phosphorylated and the 3'-hydroxylated ends of single-stranded DNA or RNA. T4 RNA Ligase 1, however, can catalyze such reactions in the presence of ATP. Therefore, this product is particularly suitable for small RNA library preparation either for miRNA sequencing or directional mRNA-Seq, to improve library quality by reducing the background of ligation reactions effectively. Please refer to Figure 1 for the performance of this product in ligating AppDNA with ssRNA.Figure 1. Ligation of AppDNA and ssRNA with Aladdin's T4 RNA Ligase 2, truncated KQ and Competitor T4 RNA Ligase 2, truncated KQ. The reaction products were resolved on denaturing polyacrylamide gel with 15% urea and image was taken after gel staining. As shown in this figure, this product had similar ligation efficiency to the competitor product.Please refer to Figure 2 for the performance of Aladdin's T4 RNA Ligase 2, truncated KQ and T4 RNA Ligase 2, truncated in deadenylating AppDNA.Figure 2. The deadenylation of AppDNA with Aladdin's T4 RNA Ligase 2, truncated KQ and T4 RNA Ligase 2, truncated . The reaction products were resolved on denaturing polyacrylamide gel with 15% urea and image was taken after gel staining. As shown in the figure, this product has similar catalytic activity to T4 RNA Ligase 2, truncated, but has no deadenylation activity on AppDNA. T4 RNA Ligase 2, truncated can deadenylate AppDNA into pDNA.sApplication：Ligation of 5'-preadenylated single-stranded DNA or RNA to the 3'-hydroxylated ssRNA; ligation of 5'-preadenylated single-stranded oligonucleotides to small RNAs for cDNA library preparations; ligation of 5'-preadenylated single-stranded oligonucleotides to RNAs for strand-specific cDNA library preparations; ligation of adapter to the 3' end of miRNA for miRNA library preparations for next generation sequencing; cDNA library preparation for small RNA transcriptome analysisSource: Truncated form of T4 RNA ligase 2 (1-249 amino acids) with R55K and K227Q double mutations, recombinantly expressed and purified from Escherichia coli.Definition of enzyme activity unit: 200 units is defined as the amount of enzyme required to give 80% ligation of a 31-mer RNA to the pre-adenylated end of a 17-mer DNA ('s Universal miRNA Cloning Linker, R0702) in a total reaction volume of 20µl in 1 hour at 25℃.Purity: Free of RNase, protease, DNA endonuclease and exonuclease.Enzyme storage buffer: 10mM Tris-HCl (pH7.5), 100mM NaCl, 0.1mM EDTA, 0.1mM DTT, 50% (v/v) Glycerol.Inactivation or inhibition: T4 RNA Ligase 2, truncated KQ can be inactivated by heating at 65℃ for 20 min or inhibited by proteinase K or EDTA.Source：Truncated form of T4 RNA ligase 2 (1-249 amino acids) with R55K and K227Q double mutations, recombinantly expressed and purified from Escherichia coli.Definition of enzyme activity unit: 200 units is defined as the amount of enzyme required to give 80% ligation of a 31-mer RNA to the pre-adenylated end of a 17-mer DNA (time's Universal miRNA Cloning Linker, R0702) in a total reaction volume of 20µl in 1 hour at 25℃.Purity: Free of RNase, protease, DNA endonuclease and exonuclease.Enzyme storage buffer：10mM Tris-HCl (pH7.5), 100mM NaCl, 0.1mM EDTA, 0.1mM DTT, 50% (v/v) glycerol.Inactivation or inhibition：T4 RNA Ligase 2, truncated KQ can be inactivated by heating at 65℃ for 20 min or inhibited by proteinase K or EDTA.Precautions：The reagents provided in this product are formulated with nuclease-free water and can be used directly for ligation reactions.Additional reagents such as RNase inhibitor and DEPC-treated Water may be required but not supplied.When using this product to ligate ssRNA and AppDNA, supplement of PEG8000 at a final concentration of 10%-30% can significantly improve the ligation efficiency.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.Instructions for Use：1. Set up the following reaction in a nuclease-free microfuge tube on ice.Reagent Volume Final Concentration ssRNA xµl 0.5 or 1µM DEPC-treated Water (4-x-y)µl -Universal miRNA Cloning Linker (, R0702) yµl 1 or 0.5 or 2 or 1µM PEG8000 (50%, RNase-free) 12µl 30% 10X T4 Rnl2tr KQ Buffer 2µl 1X RNase Inhibitor (40U/μl) 1µl 2U/μl T4 RNA Ligase2, truncated KQ (200U/µl) 1µl 10U/µl Total Volume 20µl -Note: a. As RNA manipulation is involved, RNase contamination must be avoided. Reagents and consumables required should be treated with DEPC to remove RNase. To avoid degradation of ssRNA or its ligation products, addition of RNase Inhibitor (, R0102) is recommended. b. For precious samples with limited amount of RNA, the molar ratio of the adapter and the sample RNA can be set to 2:1 to ensure complete ligation of sample RNA. However, in cases where sample RNA is sufficient, the molar ratio of the adapter and the sample RNA can be set to 1:1 or even 1:2 to save adapters. The amount of ssRNA can be appropriately adjusted according to the sample volume, and the amount of linker can be adjusted proportionally. c. When multiple reactions are required, prepare a master mix including all reagents except the substrate RNA and then dispense to different nuclease-free tubes. Finally, add the substrate RNA into each tube.2. Incubate at 25℃ for 60 min. The incubation time can be extended appropriately to make the ligation more sufficient. 3. Terminate the reaction by heating at 65℃ for 20 min.

Specification: Free of RNase, protease, and DNA endonuclease and exonuclease.

UPC:

12352204

Condition:

New

Weight:

0.07 Ounces

HazmatClass:

No

Quantity:

1

Unit of Measurement:

EA

WeightUOM:

LB

MPN:

T750875-5KU

Product Size:

5KU