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  • VentR® (exo-) DNA Polymerase

    Description

    VentR (exo-) DNA Polymerase has been genetically engineered to eliminate the 3´→ 5´ proofreading exonuclease activity associated with VentR DNA Polymerase (1). This is the preferred form for high-temperature dideoxy sequencing reactions and for high yield primer extension reactions. The fidelity of polymerization by this form is reduced to a level about 2-fold higher than that of Taq DNA Polymerase (2,3).

    Highlights

    • Isolated from a recombinant source
    • Supplied with 10X Reaction Buffer and MgSO4
    • High Thermostability: half-life of 6.7 hours at 95°C

    Product Source

    An E. coli strain that carries the Vent (D141A / E143A) DNA Polymerase gene, a genetically engineered form of the native DNA polymerase from Thermococcus litoralis (4). The native organism is capable of growth at up to 98°C and was isolated from a submarine thermal vent (5).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Magnesium Sulfate (MgSO4) Solution-20100 mM
    ThermoPol® Reaction Buffer-2010X

    Advantages and Features

    Applications

    • PCR
    • Primer extension
    • Thermal cycle sequencing
    • High temperature dideoxy-sequencing

    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
    0.1% Triton® X-100
    50% Glycerol
    pH 7.4 @ 25°C

    Heat Inactivation

    No

    Molecular Weight

    Theoretical: 89000 daltons

    5' - 3' Exonuclease

    No

    3' - 5' Exonuclease

    No

    Strand Displacement

    +++

    Resulting Ends

    • Mix of blunt and Single-base 3´ Overhangs

    Unit Assay Conditions

    1X ThermoPol Reaction Buffer, 200 µM each dNTP including [3H]-dTTP, 200 µg/ml activated calf thymus DNA.

    Error Rate

    < 190x10-6bases

    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.
    • Protein Purity (SDS-PAGE):
      The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.
    • 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. BSA is not provided with this enzyme since its presence is not necessary for most primer extension reactions. However, it is available as NEB #B9001. Acetylated BSA should not be used for primer extension reactions.
    2. Diluent D is also available. This diluent is recommended for making dilutions of VentR (exo-) Polymerase.
    3. Additional ThermoPol Reaction Buffer Packs for this product are also available. Each buffer pack contains 4 vials of 10X buffer (1.5 ml each) and 1 vial of 100 mM MgSO4.

    References

    1. Kong, H.M., Kucera, R.B. and Jack, W.E. (1993). J. Biol. Chem.. 268, 1965-1975.
    2. Mattila, P. et al. (1991). NAR. 19, 4967-4973.
    3. Eckert, K.A. and Kunkel, T.A. et al. (1991). PCR Methods and Applications 1. 17-24.
    4. Perler, F.B. et al. (1992). PNAS USA. 89, 5577-5581.
    5. Belkin, S. and Jannasch, H.W. (1985). Arch Microbiol. 141, 181-186.

    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 Vent (exo-) DNA Polymerase be used in other buffers?
    2. Are the DNA fragments produced by Vent (exo-) DNA Polymerase blunt-ended or do they have the single-base 3' overhang that Taq DNA Polymerase yields?
    3. I can't get Vent (exo-) DNA Polymerase to work yet Taq DNA Polymerase works fine.
    4. Does Bst DNA Polymerase, Large Fragment incorporate dUTP?
    5. Does Bst DNA polymerase have reverse transcriptase activity?
    6. Which thermophilic DNA polymerase should I use?
    7. What should I take into consideration when designing a set of PCR primers?
    8. How can I facilitate the amplification of templates with hairpin-loop structures or high GC-content?
    1. Guidelines for PCR Optimization for Vent DNA Polymerase
    2. Protocol for a Routine Vent (exo-) PCR

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