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  • T4 RNA Ligase 1 (ssRNA Ligase), High Concentration

    Description

    T4 RNA Ligase 1, High Concentration (M0437M), is a 3X higher concentration than our standard T4 RNA Ligase 1 (M0204S/L). This product is packaged with a vial of 100 mM ATP (N0451A). The increased concentration of reaction components is helpful in lower volume reactions where reagent volumes are limited.

    T4 RNA Ligase 1 catalyzes the ligation of a 5´ phosphoryl-terminated nucleic acid donor to a 3´ hydroxyl-terminated nucleic acceptor through the formation of a 3´ → 5´ phosphodiester bond, with hydrolysis of ATP to AMP and PPi. Substrates include singlestranded RNA and DNA as well as dinucleoside pyrophosphates (1).

    Product Source

    An E. coli strain that carries the T4 RNA Ligase 1 gene

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    T4 RNA Ligase Reaction Buffer -2010X
    PEG 8000-2050%
    Adenosine-5'-Triphosphate (ATP)-20100 mM

    Advantages and Features

    Applications

    • Ligation of ss-RNA and DNA
    • 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 oligodeoxyribonucleotides (6)
    • Incorporation of unnatural amino acids into proteins (7)

    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 Temperature

    -20°C

    Storage Conditions

    50 mM KCl
    10 mM Tris-HCl
    0.1 mM EDTA
    1 mM DTT
    50% Glycerol
    pH 7.5 @ 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).

    Notes

    1. Addition of DMSO to 10% (v/v) isrequired 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). In R. Wu, L. Grossman and K. Moldave(Ed.), Methods in Enzymology. Vol. 100, (pp. 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.

    FAQs

    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. What is the most common cause of ligation failure when using T4 RNA Ligase 1?
    4. Why doesn’t the new T4 RNA Ligase reaction buffer contain ATP?
    5. Will T4 RNA Ligase 1 ligate single stranded DNA?
    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?

    Protocols

    1. Ligation of a DNA or RNA oligo to single stranded RNA using T4 RNA Ligase 1
    2. Ligation of a DNA or RNA oligo to single stranded RNA using T4 RNA Ligase 1

    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.

    Citations

    • Shishkin AA, Giannoukos G, Kucukural A, Ciulla D, Busby M, Surka C, Chen J, Bhattacharyya RP, Rudy RF, Patel MM, Novod N, Hung DT, Gnirke A, Garber M, Guttman M, Livny J (2015)Simultaneous generation of many RNA-seq libraries in a single reaction Nat Methods PubMedID: 25730492, DOI: 10.1038/nmeth.3313
    • Jesse M. Engreitz, Klara Sirokman, Patrick McDonel, Alexander A. Shishkin, Christine Surka, Pamela Russell, Sharon R. Grossman, Amy Y. Chow, Mitchell Guttman, and Eric S. Lander (2014)RNA-RNA Interactions Enable Specific Targeting of Noncoding RNAs to Nascent Pre-mRNAs and Chromatin Sites Cell 159(1), 188-99. PubMedID: 25259926, DOI: 10.1016/j.cell.2014.08.018
    • Guttman M, Russell P, Ingolia NT, Weissman JS, Lander ES (2013)Ribosome profiling provides evidence that large noncoding RNAs do not encode proteins Cell 154(1), 240-51. PubMedID: 23810193, DOI: 10.1016/j.cell.2013.06.009
    • Engreitz JM, Pandya-Jones A, McDonel P, Shishkin A, Sirokman K, Surka C, Kadri S, Xing J, Goren A, Lander ES, Plath K, and Guttman M (2013)The Xist lncRNA exploits three-dimensional genome architecture to spread across the X chromosome Science 341(6147), 1237973. PubMedID: 23828888, DOI: 10.1126/science.1237973

    Quality Control

    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 (Buffer):
      The buffer is tested in a reaction containing a RNA substrate. After incubation there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.

    Safety Data Sheet

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

    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.