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Klenow Fragment (3'→5' exo-)

Catalog # Concentration Size List Price Quantity Your Price
M0212L 5000 units/ml 1000 units $409.00
$368.10
M0212M 50000 units/ml 1000 units $409.00
$368.10
M0212S 5000 units/ml 200 units $103.00
$92.70
Catalog # Size List Price Your Price
M0212L 1000 units $409.00
$368.10
M0212M 1000 units $409.00
$368.10
M0212S 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 Pol I, Large (Klenow) fragment was originally derived as a proteolytic product of E. coli DNA polymerase. It retains polymerase activity, but lacks both 5’ —> 3’ and 3' —> 5’ exonuclease activity.

  • Generates probes using random primers
  • Dideoxy sequencing
  • Random priming labeling
  • Moderate strand displacement activity
  • Not suitable for generating blunt ends
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:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  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
  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
  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
Application Features
  • Random priming labeling
  • DNA sequencing by the Sanger dideoxy method (2)
  • Second strand cDNA synthesis
  • Second strand synthesis in mutagenesis protocols (3).

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)

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

Companion Products
Materials Sold Separately
Notes
  • 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.
  • Klenow Fragment (3´→ 5´ exo-) is also active in all four NEBuffers when supplemented with dNTPs.
  • 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
  • Derbyshire, V. et al. (1988). Science. 240, 199-201.
  • Sanger, F. et al. (1977). Proc. Natl. Acad. Sci. USA. 74, 5463-5467.
  • Gubler, U. (1987). In S.L. Berger & A.R. Rimmel(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. Biol. 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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