Product Class: Other

DNA Polymerase I, Large (Klenow) Fragment
NEB2 cloned at NEB recombinant 25 75 Heat

Product Introduction

DNA Polymerase I, Large (Klenow) fragment was originally derived as a proteolytic product of E.coli DNA polymerase that retains polymerase and 3’ —> 5’ exonuclease activity

  • Removal of 3’ overhangs or fill-in of 5’ overhangs to form blunt ends
  • Lacks 5’ —> 3’ exonuclease activity
  • Generates probes using random primers
  • Second strand cDNA synthesis
Catalog # Size Concentration
M0210S 200.0 units 5000 units/ml
M0210L 1000.0 units 5000 units/ml
M0210M 1000.0 units 50000 units/ml

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Product Information

Description

Highlights

 

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.

Product Source

An E. coli strain that contains the E. coli polA gene that has had its 5'→3' exonuclease domain removed.
This product is related to the following categories:
DNA Manipulation Products,
This product can be used in the following applications:
Blunting,
DNA Sequencing,
Polymerases for DNA Manipulation,
RT-PCR & cDNA Synthesis,
PCR

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

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 25°C

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

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

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

Application Features

  • 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)

Product 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.
  2. 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.
  3. 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.
  4. 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.

Protocols, Manuals & Usage

Protocols

  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)

Usage & Guidelines

Tools & Resources

Selection Charts

Web Tools

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. What are the main causes of blunting reaction failure?
  6. Can this DNA polymerase be used in other NEBuffers for filling in or blunting overhangs?

Tech Tips

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

Quality, Safety & Legal

Quality Assurance Statement

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

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]

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]

Safety DataSheets

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

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

THERMOPOL® is a registered trademark of New England Biolabs, Inc.

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