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:

Store at (°C) Concentration

NEBuffer™ 2 -20 10X

Product Categories:

DNA Labeling Products,

DNA Manipulation Products,

Isothermal Amplification & Strand Displacement Products

Applications:

A-tailing,

Polymerases for DNA Manipulation,

DNA Sequencing
,
RT-PCR & cDNA Synthesis
,
PCR
- 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 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
  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
  
  Deoxynucleotide (dNTP) Solution Set
  
  Deoxynucleotide (dNTP) Solution Mix
  
  Monarch® Plasmid Miniprep Kit
  
  Monarch® DNA Gel Extraction Kit
  
  Monarch® PCR & DNA Cleanup Kit (5 μg)
  
  Materials Sold Separately
  
  NEBuffer 2
- Product 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.
Protocols, Manuals & Usage
- Protocols
  A-Tailing with Klenow Fragment (3'-->5' exo-)
- Usage Guidelines
  Activity of DNA Modifying Enzymes in CutSmart® Buffer
- Datacards
  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 [email protected] or fill out the Technical Support Form for appropriate document.)
  
  M0212Datasheet-Lot0351209
  
  M0212Datasheet-Lot0351309
  
  M0212Datasheet-Lot0351409
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 CutSmart?
  
  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?
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
  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
- 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
- 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
- Datacards
  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 [email protected] or fill out the Technical Support Form for appropriate document.)
  
  M0212Datasheet-Lot0351209
  
  M0212Datasheet-Lot0351309
  
  M0212Datasheet-Lot0351409
- 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 contact NEB's Global Business Development team at [email protected].
  
  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.
  Trademarks
  THERMOPOL^®is a registered trademark of New England Biolabs, Inc.

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Klenow Fragment (3'→5' exo-)

Highlights

Product Source

Reagents Supplied

Application Features

Unit Definition

Reaction Conditions

Storage Temperature

Storage Conditions

Heat Inactivation

Molecular Weight

5' - 3' Exonuclease

3' - 5' Exonuclease

Strand Displacement

Unit Assay Conditions

Error Rate

Companion Products

Materials Sold Separately

Trademarks

Other Products You May Be Interested In

NEB® 5-alpha Competent E. coli (High Efficiency)

Monarch® PCR & DNA Cleanup Kit (5 μg)

Blunt/TA Ligase Master Mix

	Store at (°C)	Concentration
NEBuffer™ 2	-20	10X

Klenow Fragment (3'→5' exo-)

Highlights

Product Source

Reagents Supplied

Application Features

Unit Definition

Reaction Conditions

Storage Temperature

Storage Conditions

Heat Inactivation

Molecular Weight

5' - 3' Exonuclease

3' - 5' Exonuclease

Strand Displacement

Unit Assay Conditions

Error Rate

Companion Products

Materials Sold Separately

Trademarks

Other Products You May Be Interested In

NEB® 5-alpha Competent E. coli (High Efficiency)

Monarch® PCR & DNA Cleanup Kit (5 μg)

Blunt/TA Ligase Master Mix

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