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  • T4 RNA Ligase 1 (ssRNA Ligase)

    Description

    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 (1).

    Highlights

    • Ligation of a single-stranded RNA and DNA
    • Labeling of the 3' ends of RNA with 5'-[32P]pCp
    • Synthesis of Single-stranded Oligonucleotides
    • Supplied with 10X Reaction Buffer

    Product Source

    An E. Coli strain that carries the T4 RNA Ligase I gene

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    T4 RNA Ligase Reaction Buffer 10X
    Adenosine-5'-Triphosphate (ATP)10 mM
    PEG 800050%

    Advantages and Features

    Applications

    • 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

    Properties and 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 37°C

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

    Storage Conditions

    10 mM Tris-HCl
    50 mM KCl
    1 mM DTT
    0.1 ml EDTA
    50% Glycerol
    pH 7.4 @ 25°C

    Heat Inactivation

    65°C for 15 min

    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

    Quality Control

    Quality Assurance Statement

    • Each lot is tested for contaminating single-stranded DNA exonuclease, endonuclease, ribonuclease and phosphatase activities.

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Endonuclease Activity (Nicking):
      The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
    • Exonuclease Activity (Radioactivity Release):
      The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
    • RNase Activity (1 Hour Digestion):
      The product is tested in a reaction containing a RNA substrate.  After incubation for 1 hour there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.

    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.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.
    1. Can T4 RNA Ligase 1 be used to end label single stranded DNA or RNA?
    2. Will T4 RNA Ligase 1 ligate double stranded DNA?
    3. Will T4 RNA Ligase 1 ligate single stranded DNA?
    4. What is the most common cause of ligation failure when using T4 RNA Ligase 1?
    5. Why has the unit concentration of T4 RNA Ligase 1 changed from 20,000 Units/ml to 10,000 Units/ml?
    6. Why doesn’t the new T4 RNA Ligase reaction buffer contain ATP?
    7. Will the ligation reaction using T4 RNA Ligase 1 produce circles or duplex products?
    8. Can T4 RNA Ligase 1 be used to make single stranded RNA/DNA hybrids?
    9. Can T4 RNA Ligase 1 ligate triphosphates?
    10. Can T4 RNA Ligase 1 be used to add tails to linear duplex DNA?
    11. Can a dideoxy be used to block ligation using T4 RNA Ligase 1?
    12. Can T4 RNA Ligase 1 be used to incorporate unnatural amino acids into proteins?
    13. Will PEG improve ligation with T4 RNA Ligase 1?
    1. 3´ End Labeling of RNA using T4 RNA Ligase 1
    2. RNA Circularization using T4 RNA Ligase 1
    3. Ligation of an oligo to single stranded DNA (cDNA) using T4 RNA Ligase 1

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