Product Class: Polymerase

DNA Polymerase I, Large (Klenow) Fragment

cloned at neb recombinant NEBuffer 2 heat inactivation
Catalog #SizeConcentration
M0210S200 units5,000 units/ml
M0210L1,000 units5,000 units/ml
M0210M1,000 units50,000 units/ml

Description

Highlights

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

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.

Heat Inactivated

Yes

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.

Supporting Documents

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality control's for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
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]
The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.
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.

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