Klenow Fragment (3'-->5' exo-) | NEB (2024)

HomeDNA Modifying Enzymes and Cloning TechnologiesProductsKlenow Fragment (3'→5' exo-)

• Product Information

  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

  AnE. colistrain containing a plasmid with a fragment of theE.colipolA (D355A, E357A) gene starting at codon 324.

  This product is related to the following categories:

  DNA Labeling Products,

  DNA Manipulation Products,

  This product can be used in the following applications:

  DNA Sequencing,

  A-tailing,

  Polymerases for DNA Manipulation,

  PCR

  • Reagents Supplied

    The following reagents are supplied with this product:

    NEB #	Component Name	Component #	Stored at (°C)	Amount	Concentration
    M0212S -20 Klenow Fragment (3'→5' exo-) M0212SVIAL -20 1 x 0.04 ml 5,000 units/ml NEBuffer™ 2 B7002SVIAL -20 1 x 1.25 ml 10 X
    M0212L -20 Klenow Fragment (3'→5' exo-) M0212LVIAL -20 1 x 0.2 ml 5,000 units/ml NEBuffer™ 2 B7002SVIAL -20 1 x 1.25 ml 10 X
    M0212M -20 Klenow Fragment (3'→5' exo-) M0212MVIAL -20 1 x 0.02 ml 50,000 units/ml NEBuffer™ 2 B7002SVIAL -20 1 x 1.25 ml 10 X

  • Properties & 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)

    See Also

    DNA Polymerase I, Large (Klenow) FragmentKlenow Fragment (3'→5' exo-) | NEBDNA Polymerase I, Large (Klenow) FragmentKlenow Fragment (3'-->5' exo-) | NEB

    Storage Buffer

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

    Heat Inactivation

    75°C for 20 minutes

    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

  • Advantages and Features

    Application Features

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

  • Related Products

    Companion Products

    • Deoxynucleotide (dNTP) Solution Set
    • Deoxynucleotide (dNTP) Solution Mix
    • Monarch® Plasmid Miniprep Kit
    • Monarch® DNA Gel Extraction Kit

    Materials Sold Separately

    • NEBuffer 2

  • Product 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.

• Protocols, Manuals & Usage

  • Protocols

    1. A-Tailing with Klenow Fragment (3'→5' exo-)

  • Usage Guidelines

    • Activity of DNA Modifying Enzymes in rCutSmart™ Buffer

• Tools & Resources

  • Selection Charts

    • A-tailing Selection Chart
    • Common Applications for Exonucleases and Endonucleases
    • DNA Polymerase Selection Chart

  • Web Tools

    • NEBcloner®

• FAQs & Troubleshooting

  • FAQs

    1. Which DNA polymerase can I use to blunt DNA?
    2. Can I use a DNA polymerase to fill in 3' overhangs or remove 5' overhangs?
    3. How can I heat inactivate this DNA polymerase?
    4. Can this DNA polymerase be used in labeling and partial fill reactions?
    5. Can this DNA polymerase be used in other NEBuffers for filling in or blunting overhangs?

• Citations & Technical Literature

  • Citations

    Product Citation Tool

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• Quality, Safety & Legal

  • Quality Control Assays

    Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

  • Specifications & Change Notifications

    The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

    • M0212M_v1
    • M0212S_L_v1
    • M0212M_v2
    • M0212S_L_v2

  • Certificate of Analysis

    The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

    • M0212M_v1_0351509
    • M0212S_L_v1_0351509
    • M0212M_v1_0351603
    • M0212S_L_v1_0351603
    • M0212S_L_v1_0351608
    • M0212S_L_v1_0351503
    • M0212M_v1_0351608
    • M0212M_v1_0351703
    • M0212S_L_v1_0351703
    • M0212M_v1_0351709
    • M0212S_L_v1_0351709
    • M0212M_v1_0351803
    • M0212S_L_v1_0351803
    • M0212M_v1_10017021
    • M0212L_v1_10018714
    • M0212M_v1_10018721
    • M0212S_v1_10018719
    • M0212L_v1_10029069
    • M0212M_v1_10029432
    • M0212L_v1_10034234
    • M0212S_v1_10034369
    • M0212L_v1_10034612
    • M0212S_v1_10041846
    • M0212S_v1_10044656
    • M0212S_v1_10045792
    • M0212M_v1_10041843
    • M0212L_v1_10045791
    • M0212S_v1_10048309
    • M0212M_v1_10050374
    • M0212S_v1_10050369
    • M0212L_v1_10051082
    • M0212L_v1_10055659
    • M0212M_v1_10054193
    • M0212S_v1_10056308
    • M0212L_v1_10058065
    • M0212M_v1_10058006
    • M0212S_v1_10061408
    • M0212L_v1_10063550
    • M0212S_v1_10063557
    • M0212M_v2_10065982
    • M0212L_v2_10069550
    • M0212L_v2_10073009
    • M0212M_v2_10073012
    • M0212S_v2_10073010
    • M0212L_v2_10075647
    • M0212L_v2_10083165
    • M0212M_v2_10085113
    • M0212S_v2_10084994
    • M0212L_v2_10088157
    • M0212L_v2_10094182
    • M0212S_v2_10091536
    • M0212L_v2_10098035
    • M0212M_v2_10097440
    • M0212S_v2_10098602
    • M0212M_v2_10102441
    • M0212L_v2_10106903
    • M0212S_v2_10106902
    • M0212L_v2_10111035
    • M0212S_v2_10114921
    • M0212M_v2_10122259
    • M0212L_v2_10131912
    • M0212L_v2_10119756
    • M0212S_v2_10135768
    • M0212L_v2_10138879
    • M0212L_v2_10145961
    • M0212M_v2_10146272
    • M0212L_v2_10152517
    • M0212S_v2_10159715
    • M0212M_v2_10160172
    • M0212L_v2_10162623
    • M0212M_v2_10163997
    • M0212L_v2_10167677
    • M0212M_v2_10169282
    • M0212L_v2_10172109
    • M0212S_v2_10174172
    • M0212L_v2_10177214
    • M0212L_v2_10184720
    • M0212S_v2_10185361
    • M0212L_v2_10191591
    • M0212M_v2_10201726
    • M0212S_v2_10202690
    • M0212L_v2_10203804
    • M0212S_v2_10206296
    • M0212L_v2_10208716
    • M0212M_v2_10206925
    • M0212L_v2_10218704
    • M0212L_v2_10221933
    • M0212S_v2_10215595
    • M0212S_v2_10223545
    • M0212L_v2_10230349
    • M0212L_v2_10236035
    • M0212M_v2_10234145
    • M0212S_v2_10236860
    • M0212L_v2_10239526
    • M0212M_v2_10246039
    • M0212L_v2_10246035
    • M0212S_v2_10246038
    • M0212L_v2_10255379
    • M0212M_v2_10255828

  • Safety Data Sheets

    The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

    • Klenow Fragment (3'→5' exo-)

    • NEBuffer™ 2

  • Legal and Disclaimers

    Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

    This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

    New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

    Trademarks

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