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  • Klenow Fragment (3'→5' exo-)

    Description

    Klenow Fragment (3´→ 5´ exo-) is an N-terminal truncation of DNA Polymerase I which retains polymerase activity, but has lost the 5´→ 3´ exonuclease activity and has mutations (D355A, E357A) which abolish the 3´→ 5´ exonuclease activity (1).


    Highlights

    • Isolated from a recombinant source
    • Generates probes using random primers
    • Dideoxy sequencing
    • Supplied with 10X Reaction Buffer
    • Moderate strand displacement activity

    Product Source

    An E. coli strain containing a plasmid with a fragment of the E.coli polA (D355A, E357A) gene starting at codon 324.

    Reagents Supplied

    The following reagents are supplied with this product:

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

    Advantages and Features

    Applications

    • Random priming labeling
    • DNA sequencing by the Sanger dideoxy method (2)
    • Second strand cDNA synthesis
    • Second strand synthesis in mutagenesis protocols (3).

    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

    No

    Strand Displacement

    +++

    Unit Assay Conditions

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

    Error Rate

    ~ 100x10-6bases

    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.
    • DNase Activity (Labeled Oligo, 3' extension):
      The product is tested in a reaction containing a fluorescent  labeled double stranded oligonucleotide containing a 3' extension. The percent degradation is determined by capillary electrophoresis.
    • 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.

    Notes

    1. Klenow Fragment (3´→ 5´ exo-) is not suitable for generating blunt ends because it lacks the 3´→ 5´ exonuclease necessary to remove non-templated 3´ additions.
    2. Klenow Fragment (3´→ 5´ exo-) is also active in all four NEBuffers when supplemented with dNTPs.
    3. When Klenow Fragment (3´→ 5´ exo-) is used to sequence DNA using the dideoxy method of Sanger et al., 1 unit/5 µl reaction volume is recommended.

    References

    1. Derbyshire, V. et al. (1988). Science. 240, 199-201.
    2. Sanger, F. et al. (1977). Proc. Natl. Acad. Sci. USA. 74, 5463-5467.
    3. Gubler, U. (1987). In S.L. Berger & A.R. Rimmel(Ed.), Methods in Enzymology. 152, 330-335. San Diego: Academic Press.
    4. Bebenek, K., Joyce, C.M., Fitzgerald, M.P. and Kunkel, T.A. (1990). J. Biol. 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 Klenow Fragment (3'→5' exo-) be used in other NEBuffers? (M0212)
    2. Can Klenow Fragment (3'→5' exo-) be used to blunt DNA?
    3. Can Klenow Fragment (3'→5' exo-) be used to fill in 3' overhangs?
    4. Can Klenow Fragment (3'→5' exo-) be used to remove 5' overhangs?
    5. Can Klenow Fragment (3'→5' exo-) be heat inactivated?
    6. Can Klenow Fragment (3'→5' exo-) be used in labeling reactions and partial fill in reactions?
    7. Are NEB DNA Polymerases supplied with dNTPs?
    8. Are there other methods for making probes?
    9. What is the enzyme of choice for chewing back 3' overhangs and filling in 5' overhangs (3' recessed ends)?
    1. A-Tailing with Klenow Fragment (3'-->5' exo-)

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