Product Class: Other

T4 RNA Ligase 1 (ssRNA Ligase)
NEBU cloned at NEB recombinant 25 65 Heat

There is also a high concentration (30,000 units/ml) version of this enzyme available, T4 RNA Ligase 1 (ssRNA Ligase), High Concentration (NEB #M0437M).

Product Introduction

T4 RNA Ligase 1 catalyzes the ligation of a 5´ phosphoryl-terminated nucleic acid donor to a 3´ hydroxyl-terminated nucleic acid acceptor through the formation of a 3´ → 5´ phosphodiester bond with hydrolysis of ATP to AMP and PPi. Substrates include single-stranded RNA and DNA as well as dinucleoside pyrophosphates.
Catalog # Size Concentration
M0204S 1000.0 units 10000 units/ml
M0204L 5000.0 units 10000 units/ml

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Product Information

Description

 

Product Source

An E. Coli strain that carries the T4 RNA Ligase I gene
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
  • M0204S     -20    
      T4 RNA Ligase 1 (ssRNA Ligase) M0204SVIAL -20 1 x 0.1 ml 10,000 units/ml
      T4 RNA Ligase Reaction Buffer B0216SVIAL -20 1 x 1.5 ml 10 X
      Adenosine-5'-Triphosphate (ATP) B0756AVIAL -20 1 x 0.2 ml 10 mM
      PEG 8000 B1004SVIAL -20 1 x 1 ml 1 X

Properties & Usage

Unit Definition

One unit is defined as the amount of enzyme required to convert 1 nanomole of 5´-[32P]rA16 into a phosphatase-resistant form in 30 minutes at 37°C.

Reaction Conditions

1X T4 RNA Ligase Reaction Buffer 
Supplement with 1 mM ATP
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
50 mM KCl
1 mM DTT
0.1 mM EDTA
50% Glycerol
pH 7.4 @ 25°C

Heat Inactivation

65°C for 15 minutes

Unit Assay Conditions

1X T4 RNA Ligase reaction buffer, supplemented with 1 mM ATP, is mixed with the RNA substrate (10µM of 5´-[32P]rA16 ) and varying amounts of enzyme. Incubation is at 37°C for 15 minutes (8).

Concentration:
10,000 units/ml

Application Features

  • Labeling of 3'-termini of RNA with 5'-[32P] pCp (3)
  • Inter- and intramolecular joining of RNA and DNA molecules (4,5)
  • Synthesis of single-stranded oligodeoxyribo-nucleotides (6)
  • Incorporation of unnatural amino acids into proteins (7)
  • Ligation of ss-RNA and DNA

Product Notes

  1. Addition of DMSO to 10% (v/v) is required for pCp ligation (3).

References

  1. England, T., Gumport, R. and Uhlenbeck, O. (1977). Proc. Natl. Acad. Sci. USA. 74, 4839-4842.
  2. Rand, K.N. and Gait, M.J. (1984). EMBO J.. 3, 397-402.
  3. England, T. and Uhlenbeck, O. (1978). Nature. 275, 560-562.
  4. Romaniuk, P. and Uhlenbeck, O. (1983). R. Wu, L. Grossman and K. Moldave(Ed.), Methods Enzymol.. 100, 52-56. New York: Academic Press.
  5. Moore, M.J. and Sharp, P.A. (1992). Science. 256, 992-997.
  6. Tessier, D.C., Brousseau, R., and Vernet, T. (1986). Anal. Biochem.. 158, 171-178.
  7. Noren, C.J. et al. (1989). Science. 244, 182-188.
  8. Silber, R., Malathi, B.G. and Hurwitz, J. (1972). Proc. Natl. Acad. Sci. USA. 69, 3009-3013.

Protocols, Manuals & Usage

Protocols

  1. 3´ End Labeling of RNA using T4 RNA Ligase 1
  2. RNA Circularization using T4 RNA Ligase 1(NEBM#0204)
  3. Protocol Ligation of an oligo to the 3’end of RNA using T4 RNA Ligase 1(M0204)

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. What is the optimal reaction temperature and time for T4 RNA Ligase I?
  2. Can T4 RNA Ligase 1 be used to end label single stranded DNA or RNA?
  3. Will T4 RNA Ligase 1 ligate double stranded DNA?
  4. What is the most common cause of ligation failure when using T4 RNA Ligase 1?
  5. Why doesn’t the new T4 RNA Ligase reaction buffer contain ATP?
  6. Will the ligation reaction using T4 RNA Ligase 1 produce circles or duplex products?
  7. Can T4 RNA Ligase 1 be used to make single stranded RNA/DNA hybrids?
  8. Can T4 RNA Ligase 1 ligate triphosphates?
  9. Can T4 RNA Ligase 1 be used to add tails to linear duplex DNA?
  10. Can a dideoxy be used to block ligation using T4 RNA Ligase 1?
  11. Can T4 RNA Ligase 1 be used to incorporate unnatural amino acids into proteins?
  12. Will PEG improve ligation with T4 RNA Ligase 1?
  13. Will T4 RNA Ligase 1 ligate single stranded DNA?
  14. Is PEG 8000 available for purchase?

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.

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