Product Class: Other

T4 DNA Polymerase
neb31 cloned at NEB recombinant 75 Heat

We are excited to announce that all reaction buffers are now BSA-free. NEB began switching our BSA-containing reaction buffers in April 2021 to buffers containing Recombinant Albumin (rAlbumin) for restriction enzymes and some DNA modifying enzymes. Find more details at www.neb.com/BSA-free.

Product Introduction

T4 DNA Polymerase catalyzes the synthesis of DNA in the 5´→ 3´ direction and requires the presence of template and primer. 

  • Gap filling (no strand displacement activity)
  • Removal of 3’ overhangs or fill-in of 5’ overhangs to form blunt ends
  • Lacks 5’ —> 3’ exonuclease activity
  • Probe labeling using replacement synthesis
  • Singe-strand deletion subcloning
Catalog # Size Concentration
M0203S 150 units 3000 units/ml
M0203L 750 units 3000 units/ml

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Product Information

Description

T4 DNA Polymerase catalyzes the synthesis of DNA in the 5´→ 3´ direction and requires the presence of template and primer. This enzyme has a 3´→ 5´ exonuclease activity which is much more active than that found in DNA Polymerase I (E. coli).  Unlike E. coli DNA Polymerase I, T4 DNA Polymerase does not have a 5´→ 3´ exonuclease function.

Product Source

Purified from a strain of E. coli that carries the T4 DNA Polymerase gene.
This product is related to the following categories:
DNA Manipulation
This product can be used in the following applications:
Blunting,
Polymerases for DNA Manipulation,
PCR

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

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 (5).

Reaction Conditions

1X NEBuffer™ r2.1

1X NEBuffer™ r2.1
50 mM NaCl
10 mM Tris-HCl
10 mM MgCl2
100 µg/ml Recombinant Albumin
(pH 7.9 @ 25°C)

Activity in NEBuffers

NEBuffer™ 1: 60%
NEBuffer™ 2: 100%
NEBuffer™ 3: 100%
NEBuffer™ 4: 100%

Storage Buffer

100 mM KPO4
1 mM DTT
50% Glycerol
pH 6.5 @ 25°C

Heat Inactivation

75°C for 20 minutes

Molecular Weight

Theoretical: 104000 daltons

5' - 3' Exonuclease

No

3' - 5' Exonuclease

Yes

Strand Displacement

No

Unit Assay Conditions

1X NEBuffer 2.1, 33 µM dNTPs including [3H]-dTTP, 70 µg/ml denatured herring sperm DNA.

Error Rate

~ 1x10-6bases

Application Features

  • 3´ overhang removal to form blunt ends (1,2).
  • 5´ overhang fill-in to form blunt ends (1,2).
  • Single strand deletion subcloning (3).
  • Second strand synthesis in site-directed mutagenesis (4).
  • Probe labeling using replacement synthesis (1,2).

Product Notes

  1. For fill-in reactions only: T4 DNA Polymerase can be used in NEBuffers 1.1, 2.1, 3.1 and CutSmart® Buffer as well as NEBuffers 1-4 and T4 DNA Ligase Reaction Buffer.
  2. For blunting reactions requiring removal of overhangs: T4 DNA Polymerase can be used in NEBuffers 1.1, 2.1, and CutSmart Buffer as well as NEBuffers 1, 2, and 4 and T4 DNA Ligase Reaction Buffer. NEBuffers 3.1 and 3 are not recommended when overhang removal is required.
  3. Optimal activity is observed in NEBuffer 2.1.
  4. Supplement with dNTPs.*
  5. BSA supplementation is recommended when using a buffer that does not already contain BSA.
  6. Incubate at temperature suggested for specific protocol.

    * Refer to specific protocol to determine recommended dNTP concentrations.
    Refer
  7. 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.

References

  1. Tabor, S. and Struhl, K. (1989). DNA-Dependent DNA Polymerases. In F. M. Ausebel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith and K. Struhl(Ed.), Current Protocols in Molecular Biology. 3.5.10-3.5.12. New York: John Wiley & Sons, Inc.
  2. Sambrook, J. et al. (1989). Molecular Cloning: A Laboratory Manual. (2nd ed.), 5.44-5.47. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
  3. Dale, R. et al. (1985). Plasmid. 13, 31-40.
  4. Kunkel, T.A. et al. (1987). Methods Enzymol.. 154, 367-382.
  5. Panet, A. et al. (1973). Biochemistry. 12, 5045-5050.

Protocols, Manuals & Usage

Protocols

  1. Protocol for blunting ends by 3’ overhang removal and 3’ recessed (5’ overhang) end fill-in using T4 DNA Polymerase (M0203)

Usage & Guidelines

Tools & Resources

Selection Charts

Web Tools

FAQs & Troubleshooting

FAQs

  1. Which DNA polymerase can I use to blunt DNA?
  2. Can I use a DNA polymerase to fill in 3' overhangs or remove 5' overhangs?
  3. How can I heat inactivate this DNA polymerase?
  4. Can this DNA polymerase be used in labeling and partial fill reactions?
  5. What are the main causes of blunting reaction failure?
  6. Can this DNA polymerase be used in other NEBuffers for filling in or blunting overhangs?

Tech Tips

Excess enzyme, temperatures above 12°C, or limited dNTP concentrations can cause excessive 3’ → 5’ exonuclease degradation, which eliminates blunt end formation.

Quality, Safety & Legal

Quality Assurance Statement

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 DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email 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.

Licenses

THERMOPOL® is a registered trademark of New England Biolabs, Inc.

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