Product Class: Other

T4 RNA Ligase 2, truncated K227Q
NEBU recombinant 25 65 Heat

Product Introduction

T4 RNA Ligase 2, truncated K227Q ligates the pre-adenylated 5´ end of DNA or RNA to the 3´ end of RNA.

  • Reduced lysyl adenylation activity
  • Isolated from a recombinant source
  • Supplied with 10X Reaction Buffer
  • Requires 5' pre-adenylated RNA or DNA for ligation
  • Tested for the absence of endonucleases, exonucleases, RNases and phosphatases
  • Not sure which ligase to choose? Refer to our DNA and RNA Ligase Properties Chart
Catalog # Size Concentration
M0351S 2000.0 units 200000 units/ml
M0351L 10000.0 units 200000 units/ml

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Product Information

Description

T4 RNA Ligase 2, truncated K227Q (T4 Rnl2tr K227Q) specifically ligates the pre-adenylated 5´ end of DNA or RNA to the 3´ OH end of RNA. The enzyme does not use ATP for ligation but requires pre-adenylated linkers. 

T4 Rnl2tr K227Q is a point mutant of T4 RNA Ligase 2, truncated (NEB# M0242) . Mutation of K227 in T4 RNA Ligase 2 reduces enzyme lysyl adenylation (1). This mutation further reduces the formation of undesired ligation products (concatemers and circles) by T4 Rnl2tr (2), possibly by reducing the trace activity of T4 Rnl2tr in transfer of adenylyl groups from linkers to the 5´-phosphates of input RNAs. 

The exclusion of ATP, use of pre-adenylated linkers, and the reduced enzyme lysyl adenylation activity provide the lowest possible background in ligation reactions. This enzyme has been used for optimized linker ligation for the cloning of microRNAs (2).

Product Source

T4 Rnl2tr K227Q is expressed as an MBP fusion from a plasmid in E. coli which encodes the first 249 amino acids of the full length T4 RNA Ligase 2 with a lysine to glutamine mutation at position 227.
This product is related to the following categories:
RNA Ligation

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

Properties & Usage

Unit Definition

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 Universal miRNA Cloning Linker (NEB #S1315 ) in a total reaction volume of 20 µl in 1 hour at 25°C. 

5´-FAM- rArGrUrCrGrUrArGrCrCrUrUrUrArUrCrCrGrArGrArUrUrCrArGrCrArArUrA-3´ 

5´-rAppCTGTAGGCACCATCAAT–NH2-3´ 

Molarity:
14 µM

Concentration:
200,000 units/ml

Reaction Conditions

1X T4 RNA Ligase Reaction Buffer 
Incubate at 25°C

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

Storage Buffer

10 mM Tris-HCl
100 mM NaCl
1 mM DTT
0.1 mM EDTA
50% Glycerol
pH 7.5 @ 25°C

Heat Inactivation

65°C for 20 minutes

Molecular Weight

Theoretical: 71406.97 daltons

Specific Activity

200,000 units/mg

Unit Assay Conditions

1X T4 RNA Ligase Reaction Buffer supplemented to 10% (w/v) PEG MW 8000, 20 pmol of 5´-FAM labeled RNA, and 40 pmol preadenylated DNA linker. After incubation at 25°C for 1 hour, the ligated product is detected on a 15% denaturing polyacrylamide gel.

Application Features

  • Ligate a pre-adenylated DNA or RNA sequence tag to any RNA 3´-end
  • Join a single stranded adenylated primer to small RNAs for cDNA library creation
  • Join a single stranded adenylated primer to RNA for strand-specific cDNA library construction

Product Notes

  1. In our tests, this ligase can be used interchangeably with T4 Rnl2tr (NEB #M0242), but may require increased incubation time. Ligation activity is stimulated by adding PEG (See FAQ).

References

  1. Yin, S., Ho, C.K. and Shuman S. (2003). J. Biol. Chem. 278, 17601-17608.
  2. Hafner, M., et al. (2008). Methods. 44, 3-12.
  3. Viollet, S., et al. (2011). BMC Biotechnology. 11, 72.

Protocols, Manuals & Usage

Protocols

  1. Protocol for ligation of the 3´ OH of RNA to the 5´ pre-adenylated DNA with T4 RNA Ligase 2 truncated K227Q (NEB #M0351)

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. The molecular weight of T4 Rnl2tr K227Q is 71.6 kDa. Is it a fusion?
  2. Can T4 Rnl2tr K227Q be used in other NEBuffers?
  3. Are there differences in ligation efficiency for T4 Rnl2tr and T4 Rnl2tr K227Q?
  4. What can T4 Rnl2tr K227Q ligate?
  5. Does PEG stimulate ligation efficiency?
  6. How can I increase ligation efficiency?
  7. Is PEG 8000 available for purchase?

Tech Tips

Compared to T4Rnl2tr truncated, this mutant produces less side products.

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email [email protected].

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

Trademarks

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