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  • Bsu DNA Polymerase, Large Fragment

    Description

    Bsu DNA Polymerase I, Large Fragment retains the 5´→ 3´ polymerase activity of the Bacillus subtilis DNA polymerase I (1), but lacks the 5´→ 3´ exonuclease domain. This large fragment naturally lacks 3´→ 5´ exonuclease activity.


    Product Source

    An E. coli strain that contains a genetic fusion of the Bacillus subtilis DNA polymerase I gene (starting from codon 297 thus lacking the 5´→ 3´ exonuclease domain), and the gene coding for maltose binding protein (MBP). The fusion protein is purified to near homogeneity and the MBP portion is cleaved off in vitro. The remaining DNA polymerase is purified free of MBP.

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBuffer 2-2010X

    Advantages and Features

    Applications

    • Random primer labeling
    • Second strand cDNA synthesis
    • Single dA tailing
    • Strand displacement DNA synthesis (2)

    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 37°C.

    Reaction Conditions

    1X NEBuffer 2
    Incubate at 37°C

    1X NEBuffer 2:
    50 mM NaCl
    10 mM Tris-HCl
    10 mM MgCl2
    1 mM DTT
    pH 7.9 @ 25°C

    Storage Temperature

    -20°C

    Heat Inactivation

    75°C for 20 min

    5' - 3' Exonuclease

    No

    3' - 5' Exonuclease

    No

    Strand Displacement

    +

    Unit Assay Conditions

    1X NEBuffer 2, 33 µM dNTPs including [3H]-dTTP and 70 µg/ml denatured herring sperm DNA.

    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.
    • Single Stranded DNase Activity (FAM Labeled Oligo):
      The product is tested in a reaction containing a fluorescent internal labeled single stranded oligonucleotide. The percent degradation is determined by capillary electrophoresis.

    Notes

    1. Bsu DNA Polymerase, Large Fragment is not suitable for generating blunt ends because it lacks the 3´→ 5´ exonuclease necessary to remove non-templated 3´ additions.
    2. Bsu DNA Polymerase, Large Fragment retains 50% activity at 25°C and is twice as active as Klenow Fragment (3´→ 5´ exo–) at this temperature.

    References

    1. Okazaki, T. et al. (1964). J. Biol. Chem.. 239, 259-268.
    2. Piepenburg, O. et al. (2006). PLOS Biology. 4, 1115-1121.

    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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