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Home DNA Modifying Enzymes and Cloning Technologies Products Klenow Fragment (3'→5' exo-)

Klenow Fragment (3'→5' exo-)

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

Catalog #	Concentration	Size
M0212S	5,000 units/ml	200 units
M0212L	5,000 units/ml	1,000 units
M0212M	50,000 units/ml	1,000 units
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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

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:

Show all

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

This product is related to the following categories:: DNA Labeling Products,; DNA Manipulation Products,; Isothermal Amplification & Strand Displacement Products

This product can be used in the following applications:: Strand Displacement Amplification & Nicking Enzyme,; A-tailing,; DNA Sequencing,; Polymerases for DNA Manipulation,; RT-PCR & cDNA Synthesis,

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).
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 min
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
Related Products
Companion Products
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
  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
  Can Klenow Fragment (3'→5' exo-) be used in other NEBuffers, including rCutSmart?
  
  Can Klenow Fragment (3'→5' exo-) be used to blunt DNA?
  
  Can Klenow Fragment (3'→5' exo-) be used to fill in 3' overhangs?
  
  Can Klenow Fragment (3'→5' exo-) be used to remove 5' overhangs?
  
  Can Klenow Fragment (3'→5' exo-) be heat inactivated?
  
  Can Klenow Fragment (3'→5' exo-) be used in labeling reactions and partial fill in reactions?
  
  Are there other methods for making probes?
  
  What is the enzyme of choice for chewing back 3' overhangs and filling in 5' overhangs (3' recessed ends)?
  
  Are NEB DNA Polymerases supplied with dNTPs?
Citations & Technical Literature
- Citations
  
  Product Citation Tool
  
  Powered by Bioz See more details on Bioz
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
  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
- 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
  
  This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).
  
  While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.
  
  For more information about commercial rights, please email us at 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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