T4 DNA PolymeraseProduct information
- Extreme fidelity (6)
- Gap filling (no strand displacement activity)
- Best enzyme for creating blunt ends
- Isolated from a recombinant source
Product SourcePurified from a strain of E. coli that carries the T4 DNA Polymerase gene.
The following reagents are supplied with this product:
|Store at (°C)||Concentration|
Advantages and 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).
Properties and Usage
Unit DefinitionOne 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).
1X NEBuffer 2.1
Incubate at 12°C
1X NEBuffer 2.1:
50 mM NaCl
10 mM Tris-HCl
10 mM MgCl2
100 μg/ml BSA
pH 7.9 @ 25°C
100 mM KPO4
1 mM DTT
pH 6.5 @ 25°C
Heat Inactivation75°C for 20 min
Molecular WeightTheoretical: 104000 daltons
5' - 3' ExonucleaseNo
3' - 5' ExonucleaseYes
Unit Assay Conditions1X NEBuffer 2.1, 33 µM dNTPs including [3H]-dTTP, 70 µg/ml denatured herring sperm DNA.
Quality Control AssaysThe following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
- Endonuclease Activity (Nicking):
The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
LicensesTHERMOPOL® is a registered trademark of New England Biolabs, Inc.
- 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.
- 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.
- Optimal activity is observed in NEBuffer 2.1.
- Supplement with dNTPs.*
- BSA supplementation is recommended when using a buffer that does not already contain BSA.
- Incubate at temperature suggested for specific protocol.
*Refer to specific protocol to determine recommended dNTP concentrations.
- 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.
- Sambrook, J. et al. (1989). Molecular Cloning: A Laboratory Manual. (2nd ed.), 5.44-5.47. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
- Dale, R. et al. (1985). Plasmid. 13, 31-40.
- Kunkel, T.A. et al. (1987). Methods Enzymol.. 154, 367-382.
- Panet, A. et al. (1973). Biochemistry. 12, 5045-5050.
- Can T4 DNA Polymerase be used in other NEBuffers?
- Can T4 DNA Polymerase be used to blunt DNA?
- Can T4 DNA Polymerase be used to fill in 3' overhangs?
- Can T4 DNA Polymerase be used to remove 5' overhangs?
- Can T4 DNA Polymerase be heat inactivated?
- Are the nucleotides needed to remove a 3' overhang using T4 DNA Polymerase?
- What are the main causes of blunting reaction failure using T4 DNA Polymerase?
- Are NEB DNA Polymerases supplied with dNTPs?
- Can T4 DNA Polymerase be used in labeling reactions and partial fill in reactions?
- Is T4 DNA Polymerase active at room temperature?
- Is T4 DNA Polymerase the enzyme of choice for removing 3' overhangs and filling in 5' overhangs (3' recessed ends)?