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  • Therminator™ II DNA Polymerase

    Discontinued Date

    01/01/2013
    cloned at neb recombinant unique buffer heat inactivation no
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    Discontinued Products

    Description

    Therminator II DNA Polymerase is a 9°N™ DNA Polymerase variant with an enhanced ability to incorporate modified substrates such as dideoxynucleotides, ribonucleotides and acyclonucleotides (1,2).

    Therminator II DNA Polymerase was derived from Therminator DNA Polymerase and differs in having one additional amino acid change. This change allows more efficient incorporation of ribonucleotides (1) and nucleotides with modified 3´ functional groups (3).

    Highlights

    • Enhanced incorporation of modified nucleotides, especially ribonucleotides
    • Isolated from recombinant source
    • Supplied with 10X Reaction Buffer

    Product Source

    An E. coli strain that carries the 9°N (D141A / E143A / Y409V / A485L) DNA Polymerase gene, a genetically engineered form of the native DNA polymerase from Thermococcus species 9°N-7.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    ThermoPol® Reaction Buffer-2010X
    BSA-20100X

    Advantages and Features

    Applications

    • DNA sequencing by partial ribosubstitution (4)
    • DNA sequencing using dideoxy (5) or acyclo (6) chain terminators
    • SNP analysis with dideoxy or acyclo chain terminators (7)

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in a total reaction volume in 30 minutes at 75°C.

    Reaction Conditions

    1X ThermoPol® Reaction Buffer

    1X ThermoPol® Reaction Buffer:
    20 mM Tris-HCl
    10 mM (NH4)2SO4
    10 mM KCl
    2 mM MgSO4
    0.1% Triton® X-100
    pH 8.8 @ 25°C

    Storage Temperature

    -20°C

    Storage Conditions

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

    Heat Inactivation

    No

    5' - 3' Exonuclease

    No

    3' - 5' Exonuclease

    No

    Strand Displacement

    +

    Unit Assay Conditions

    1X ThermoPol Reaction Buffer, 200 µM dNTPs including [3H]-dTTP and 15 nM primed single-stranded M13mp18.

    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.

    References

    1. Gardner, A.F. and Jack, W.E. (1999). Nucleic Acids Research. 27, 2545-2555.
    2. Gardner, A.F. and Jack, W.E. (2002). Nucleic Acids Research. 30, 605-613.
    3. Ruparel, H. et al. (2005). Proc. Natl. Acad. Sci., USA. 26, 5932-5937.
    4. Barnes, W.F. (1978). J. Mol. Biol.. 119, 83-99.
    5. Sanger, F., Nicklen, S. and Coulson, A.R. (1977). Proc. Natl. Acad. Sci., USA. 74, 5463-5467.
    6. Trainor, G.L. (1996). U.S. Patent 5,558,991.
    7. Haff, L.A. and Simirnov, I.P. (1997). Genome Methods. 7, 378-388.

    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.

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