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  • DNA Polymerase I, Large (Klenow) Fragment

    Description

    DNA Polymerase I, Large (Klenow) Fragment is a proteolytic product of E. coli DNA Polymerase I which retains polymerization and 3'→ 5' exonuclease activity, but has lost 5'→ 3' exonuclease activity (1). Klenow retains the polymerization fidelity of the holoenzyme without degrading 5' termini.

    Highlights

    • Isolated from a recombinant source
    • Dideoxy sequencing
    • Generates probes using random primers
    • Creates blunt ends
    • Supplied with 10X Reaction Buffer

    Product Source

    Purified from a strain of E. coli that carries the DNA Polymerase I, Large (Klenow) Fragment gene.

    Reagents Supplied

    The following reagents are supplied with this product:

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

    Advantages and Features

    Applications

    • DNA sequencing by the Sanger dideoxy method (2)
    • Fill-in of 5´ overhangs to form blunt ends (3)
    • Removal of 3´ overhangs to form blunt ends (3)
    • Second strand cDNA synthesis
    • Second strand synthesis in mutagenesis protocols (4).

    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

    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

    Storage Conditions

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

    Heat Inactivation

    75°C for 20 min

    Molecular Weight

    Theoretical: 68000 daltons

    5' - 3' Exonuclease

    No

    3' - 5' Exonuclease

    Yes

    Strand Displacement

    +

    Unit Assay Conditions

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

    Error Rate

    ~ 18x10-6bases

    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.

    Notes

    1. Protocol for blunting ends by 3' overhang removal and 3' recessed end fill-in: 

      • DNA should be dissolved in 1X NEBuffer 1-4 or T4 DNA Ligase Reaction Buffer supplemented with 33 μM each dNTP. Add 1 unit DNA Polymerase I, Large (Klenow) Fragment per microgram DNA and incubate 15 minutes at 25°C. Stop reaction by adding EDTA to a final concentration of 10 mM and heating at 75°C for 20 minutes. 
      • CAUTION: Elevated temperatures, excessive amounts of enzyme, failure to supplement with dNTPs or long reaction times may result in recessed ends due to the 3'→ 5' exonuclease activity of the enzyme.
    2. When DNA Polymerase I, Large (Klenow) Fragment is used to sequence DNA using the dideoxy method of Sanger et al., 1 unit/5 μl reaction volume is recommended.
    3. DNA Polymerase I, Large (Klenow) Fragment is also active in all four NEBuffers and T4 DNA Ligase Reaction Buffer when supplemented with dNTPs.

    References

    1. Jacobsen, H., Klenow, H. and Overgaard-Hansen, K. (1974). Eur. J. Biochem.. 45, 623-627.
    2. Sanger, F. et al. (1977). Proc. Natl. Acad. Sci. USA. 74, 5463-5467.
    3. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. (2nd ed.), 5.40-5.43. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
    4. Gubler, U. (1987). In S.L. Berger and A.R. Kimmel(Ed.), Methods in Enzymology. 152, 330-335. San Diego: Academic Press.
    5. Bebenek, K., Joyce, C.M., Fitzgerald, M.P. and Kunkel, T.A. (1990). J. Bio. Chem . 265, 13878-13887.

    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 DNA Polymerase I, Large (Klenow) Fragment be used in other NEBuffers?
    2. Can DNA Polymerase I, Large (Klenow) Fragment be used to blunt DNA?
    3. Can DNA Polymerase I, Large (Klenow) Fragment be used to fill in 3' overhangs?
    4. Can DNA Polymerase I, Large (Klenow) Fragment be used to chew back 5' overhangs?
    5. Are NEB DNA Polymerases supplied with dNTPs?
    6. Can DNA Polymerase I, Large (Klenow) Fragment be heat inactivated?
    7. Are the nucleotides needed to remove a 3' overhang with DNA Polymerase I, Large (Klenow) Fragment?
    8. What are the main causes for blunting reaction failure using DNA Polymerase I, Large (Klenow) Fragment?
    9. Can DNA Polymerase I, Large (Klenow) Fragment be used in labeling reactions and partial fill in reactions?
    10. Is DNA Polymerase I, Large (Klenow) Fragment the enzyme of choice for chewing back 3' overhangs and filling in 5' overhangs (3' recessed ends)?
    1. Protocol for blunting ends by 3' overhang removal and fill-in of 3' recessed (5' overhang) ends using DNA Polymerase I, Large (Klenow) Fragment (M0210)

    Selection Tools

    Excess enzyme, temperatures above 25°C, or limited dNTP concentrations can cause excessive 3’ ? 5’ exonuclease degradation, which eliminates blunt end formation.