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T4 DNA Polymerase

#EP0061 Supplied with: 5X Reaction Buffer	100 u (5 u/µl) 1 ml
#EP0062 Supplied with: 5X Reaction Buffer	500 u (5 u/µl) 5x1 ml
Related Documents (in pdf, ~40-60 KB): Certificate of Analysis: #EP0061, #EP0062 MSDS (English) MSDS (English-USA) MSDS (German)

 

Feature
Active in Fermentas buffers for restriction enzymes, PCR and RT.

Description
The 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.

Applications

• Blunting DNA with 5'- or 3'-protruding termini (1, 2), see Protocol for Blunting DNA with 5'- or 3'- protruding Termini.
• Synthesis of labeled DNA probes by the replacement reaction (3).
• Oligonucleotide-directed site-specific mutagenesis (4).
• Ligation-independent cloning of PCR fragments (5-6).

Quality Control
The absence of endodeoxyribonucleases confirmed by appropriate quality test.

Concentration
5 u/µl

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 m M Tris-HCl (pH 8.8), 6.7 m M MgCl₂, 1 m M DTT, 16.7 m M (NH₄)₂SO₄, 0.2 mg/ml BSA, 0.033 m M 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 m M potassium phosphate (pH 7.5), 200 m M KCl, 2 m M DTT and 50% (v/v) glycerol.

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

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.

Note
The 3'=>5' exonuclease activity of T4 DNA Polymerase is stronger on single-stranded than on double-stranded DNA and greater (more than 200 times) than that of DNA Polymerase I, E.coli (1).

Related Products

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

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

Updated kovo 18, 2008 10:05