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  • 9°Nm™ DNA Polymerase

    Discontinued Date

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

    Description

    9°Nm™ DNA Polymerase is a thermophilic DNA polymerase that has been genetically engineered to have a decreased 3'→ 5' proofreading exonuclease activity (1-5% of the wildtype). 9°Nm DNA Polymerase features a half-life of 6.7 hours at 95°C.

    9°Nm DNA Polymerase crystals (Ira Schildkraut and Rebecca Kucera, New England Biolabs, Inc.)

    Highlights

    • Isolated from a recombinant source
    • Supplied with 10X Reaction Buffer

    Product Source

    An E. coli strain that carries the 9°N (E143D) DNA Polymerase gene, (1,2) a genetically engineered form of the native DNA polymerase from the extremely thermophilic marine archaea Thermococcus species 9°N-7. The archaea was isolated from a submarine thermal vent, at a depth of 2,500 meters, 9° north of the equator at the East Pacific Rise (3).

    Reagents Supplied

    The following reagents are supplied with this product:

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

    Advantages and Features

    Applications

    • Primer extension
    • SNP analysis

    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 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

    Molecular Weight

    Theoretical: 90000 daltons

    5' - 3' Exonuclease

    No

    3' - 5' Exonuclease

    Yes

    Strand Displacement

    +

    Resulting Ends

    • Mix of blunt and Single-base 3´ Overhangs

    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.

    Notes

    1. It is suggested that the number of units be optimized with each primer:template.
    2. MgSO4 is usually optimal at 2 mM, although occasionally a 1 mM level is better. For such reactions, we offer a ThermoPol II (Mg-free) Reaction Buffer Pack (#B9005) to which 1 mM MgSO4 can be added.
    3. Diluent E (#B8005) is available for making dilutions of 9°Nm DNA Polymerase.
    4. Additional buffer packs are also available (#B9004). Each buffer pack contains 4 vials of 10X ThermoPol Reaction Buffer (1.5 ml each) and 1 vial of 100 mM MgSO4.

    References

    1. Southworth, M.W. et al. (1996). Proc. Natl. Acad. Sci. USA. 93, 5281-5285.
    2. Rodriquez, A.C. et al. (2000). J. Mol. Biol.. 299, 447-462.
    3. Dr. Holger Jannasch (1991). Thermococcus sp. (strain 9°N-7) isolated. Woods Hole Oceanographic Institute.

    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. I can't get 9°Nm DNA Polymerase to work, yet Taq DNA Polymerase works fine.
    2. Are the DNA fragments produced by 9°Nm DNA Polymerase blunt-ended or do they have the single-base 3' overhang that Taq DNA Polymerase yields?
    3. Can I use 9°Nm DNA Polymerase to polish the ends of DNA fragments?
    4. Can 9°Nm DNA Polymerase be used in other buffers?

    Selection Tools

    Usage Guidelines & Tips

    Troubleshooting Guides

    For reactions requiring Mg2+ optimization, ThermoPol II (Mg-Free) Reaction Buffer Packs are available (NEB# B9005S).