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Enzymes

T4 DNA Polymerase

Product information

Features

• Stronger 3’=>5’ exonuclease activity on single-stranded than on double-stranded DNA and greater (more than 200 times) than DNA polymerase I, E.coli and Klenow fragment (1).
• Active in restriction enzyme, PCR, RT and T4 DNA Ligase buffers.

Applications

• Blunting of DNA ends: fill-in 5'-overhangs or/and removal of 3'-overhangs (1, 2).
• Blunting of PCR products with 3'-dA overhangs.
• Synthesis of labeled DNA probes by the replacement reaction (3).
• Oligonucleotide-directed site-specific mutagenesis (4).
• Ligation-independent cloning of PCR products (5, 6).

Description

T4 DNA Polymerase, a template-dependent DNA polymerase, catalyzes 5'=>3' synthesis from primed single-stranded DNA. The enzyme has a 3'=>5' exonuclease activity, but lacks 5'=>3' exonuclease activity.

Source

E.coli cells with a cloned gene 43 of bacteriophage T4.

Molecular Weight

104 kDa monomer.

Definition of Activity Unit

One unit of the enzyme catalyzes the incorporation of 10 nmol of deoxyribonucleotides into a polynucleotide fraction (adsorbed on DE-81) in 30 min at 37°C.

Enzyme activity is assayed in the following mixture: 67 mM Tris-HCl (pH 8.8), 6.7 mM MgCl₂, 1 mM DTT, 16.7 mM (NH₄)₂SO₄, 0.2 mg/ml BSA, 0.033 mM of each dNTP, 0.4 MBq/ml [³H]-dTTP and 0.2 mM heat-denatured and nuclease-digested calf thymus DNA.

Storage Buffer

The enzyme is supplied in:
20 mM potassium phosphate (pH 7.5), 200 mM KCl, 2 mM DTT and 50% (v/v) glycerol.

5X Reaction Buffer

335 mM Tris-HCl (pH 8.8 at 25°C), 33 mM MgCl₂, 5 mM DTT, 84 mM (NH₄)₂SO₄.

Quality Control

The absence of endodeoxyribonucleases confirmed by appropriate quality tests.

Inhibition and Inactivation

• Inhibitors: metal chelators, nucleotide analogs 2(p-n-butylanilino)-dATP, N2-(p-n-butylphenyl)-dGTP), SH-blocking compounds (7).
• Inactivated by heating at 75°C for 10 min.

Patents, Licenses, Trademarks

Protocols & recommendations

RECOMMENDATIONS FOR USE

ADDITIONAL PROTOCOLS

Blunting of 5'- or 3'-overhangs with T4 DNA Polymerase

T4 DNA Polymerase fills-in 5'-overhangs and removes 3'-overhangs.

1. Prepare the following reaction mixture:
   

   5X reaction buffer for T4 DNA Polymerase	4 µl
   Linear DNA or PCR product	1 µg
   dNTP Mix, 2 mM each	1 µl (0.1 mM final concentration)
   T4 DNA Polymerase	0.2 µl (1 u)
   Water, nuclease-free	to 20 µl
   Total volume	to 20 µl

2. Mix thoroughly, spin briefly and incubate at 11°C for 20 min or at room temperature for 5 min.
3. Stop the reaction by heating at 75°C for 10 min.

References

1. Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, Third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.
2. Ausubel, F.M., et al., Current Protocols in Molecular Biology, vol. 1, John Wiley & Sons, Inc., Brooklyn, New York, 3.5.11-3.5.12, 1994-2005.
3. Challberg, M.D., Englund, P.T., Specific labeling of 3’-termini with T4 DNA polymerase, Methods Enzymol., 65, 39-43, 1980.
4. Kunkel, I.A., et al., Rapid and efficient site-specific mutagenesis without phenotypic selection, Methods Enzymol., 154, 367-382, 1987.
5. Haun, R.S., et al., Rapid, reliable ligation-independent cloning of PCR products using modified plasmid vectors, BioTechniques, 13, 515-518, 1992.
6. Wang, K., et al., A simple method using T4 DNA polymerase to clone polymerase chain reaction products, BioTechniques, 17, 236-238, 1994.
7. Eun, H-M., Enzymology Primer for Recombinant DNA Technology, Academic Press, Inc., 1996

Related products

• dNTP Set
• T4 DNA Ligase
• dNTP Mixes
• 0.5 M EDTA, pH 8.0
• Water, nuclease-free