T4 RNA Ligase 2, truncated K227Q

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

Bioz Badge Exists : True

Catalog #	Size	Concentration
M0351S	2000 units	200000 units/ml
M0351L	10000 units	200000 units/ml

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

This product can be used in the following applications:: RNA Cloning

Reagents Supplied

The following reagents are supplied with this product:

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

NEB #			Stored at (°C)
M0351S			-20

Component Name	Component #	Stored at (°C)	Amount	Concentration
T4 RNA Ligase 2, truncated K227Q	M0351SVIAL	-20	1 x 0.01 ml	200,000 units/ml
T4 RNA Ligase Reaction Buffer	B0216SVIAL	-20	1 x 1.5 ml	10 X
50% PEG 8000	B1004SVIAL	-20	1 x 1 ml	1 X

NEB #			Stored at (°C)
M0351L			-20

Component Name	Component #	Stored at (°C)	Amount	Concentration
T4 RNA Ligase 2, truncated K227Q	M0351LVIAL	-20	1 x 0.05 ml	200,000 units/ml
T4 RNA Ligase Reaction Buffer	B0216SVIAL	-20	1 x 1.5 ml	10 X
50% PEG 8000	B1004SVIAL	-20	1 x 1 ml	1 X

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

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

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

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

Tech Tips

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

Citations & Technical Literature

Citations

Additional Citations

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]

M0351S_L_v2

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.

T4 RNA Ligase 2, truncated K227Q

T4 RNA Ligase Reaction Buffer

PEG 8000

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 busdev@neb.com.

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

NEW ENGLAND BIOLABS^® is a registered trademark of New England Biolabs, Inc.

T4 RNA Ligase 2, truncated K227Q

Product Introduction

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