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DNA Polymerase I, Large (Klenow) Fragment

Catalog # Concentration Size List Price Quantity Your Price
M0210L 5000 units/ml 1000 units $409.00
$368.10
M0210M 50000 units/ml 1000 units $409.00
$368.10
M0210S 5000 units/ml 200 units $103.00
$92.70
Catalog # Size List Price Your Price
M0210L 1000 units $409.00
$368.10
M0210M 1000 units $409.00
$368.10
M0210S 200 units $103.00
$92.70
Catalog #
Qty:
 
*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

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
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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

 

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:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  DNA Polymerase I, Large (Klenow) Fragment M0210SVIAL -20 1 x 0.04 ml 5,000 units/ml
  NEBuffer™ 2 B7002SVIAL -20 1 x 1.25 ml 10 X
  DNA Polymerase I, Large (Klenow) Fragment M0210LVIAL -20 1 x 0.2 ml 5,000 units/ml
  NEBuffer™ 2 B7002SVIAL -20 1 x 1.25 ml 10 X
  DNA Polymerase I, Large (Klenow) Fragment M0210MVIAL -20 1 x 0.02 ml 50,000 units/ml
  NEBuffer™ 2 B7002SVIAL -20 1 x 1.25 ml 10 X
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)

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

Companion Products
Materials Sold Separately
Notes
  • 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.
  • 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.
  • 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.
  • DNA Polymerase I, Large (Klenow) Fragment is also active in all four NEBuffers and T4 DNA Ligase Reaction Buffer when supplemented with dNTPs.
References
  • Jacobsen, H., Klenow, H. and Overgaard-Hansen, K. (1974). Eur. J. Biochem.. 45, 623-627.
  • Sanger, F. et al. (1977). Proc. Natl. Acad. Sci. USA. 74, 5463-5467.
  • 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.
  • Gubler, U. (1987). In S.L. Berger and A.R. Kimmel(Ed.), Methods in Enzymology. 152, 330-335. San Diego: Academic Press.
  • Bebenek, K., Joyce, C.M., Fitzgerald, M.P. and Kunkel, T.A. (1990). J. Bio. Chem . 265, 13878-13887.
Quality Control Assay
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 Data Sheets
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