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T4 DNA Polymerase

B buffer for 100% activity FastDigest buffer for 100% activity G buffer for 100% activity O buffer for 100% activity Recombinant enzyme R buffer for 100% activity Tango buffer for 100% activity Thermal inactivation at 75°C in 10 min

T4 DNA Polymerase is template-dependent and catalyzes the 5'-3' synthesis from primed single-stranded DNA.
  
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Thermo ScientificT4 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.

Highlights

Applications

  • Blunting of DNA ends: fill-in of 5'-overhangs or/and removal of 3'-overhangs(see​ References1, 2)
  • Blunting of PCR products with 3'-dA overhangs
  • Synthesis of labeled DNA probes by the replacement reaction(see​ Reference3)
  • Oligonucleotide-directed site-specific mutagenesis(see​ Reference4)
  • Ligation-independent cloning of PCR products(see​ References5, 6)
  
Storage Condition-20 C
HazardousNo
5× Reaction Buffer335mM Tris-HCl (pH8.8 at 25°C), 33mM MgCl2, 5mM DTT, 84mM (NH4)2SO4 
Concentration5U/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 30min at 37°C.
  • Enzyme activity is assayed in the following mixture: 67mM Tris-HCl (pH 8.8), 6.7mM MgCl2, 1mM DTT, 16.7mM (NH4)2SO4, 0.2mg/mL BSA, 0.033mM of each dNTP, 0.4 MBq/mL [3H]-dTTP, and 0.2mM heat-denatured and nuclease-digested calf thymus DNA.
InactivationInactivated by heating at 75°C for 10min.
InhibitionInhibitors: metal chelators, nucleotide analogs 2(p-n-butylanilino)-dATP, N2-(p-n-butylphenyl)-dGTP), SH-blocking compounds (see Reference 7) 
Molecular Weight104kDa monomer
Quality ControlThe absence of endodeoxyribonucleases confirmed by appropriate quality tests.
SourceE.coli cells with a cloned gene 43 of bacteriophage T4
Storage BufferThe enzyme is supplied in 20mM potassium phosphate (pH7.5), 200mM KCl, 2mM DTT, and 50%(v/v) glycerol.

References

  1. J. Sambrook, D. W. Russell, Molecular Cloning: A Laboratory Manual, Third edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York 2001).
  2. F. M. Ausubel et al., Current Protocols in Molecular Biology (vol. 1, John Wiley).
  3. M. D. Challberg, P. T. Englund, Specific labeling of 3'-termini with T4 DNA polymerase.Methods Enzymol.65, 39-43 (1980).
  4. I. A. Kunkel et al., Rapid and efficient site-specific mutagenesis without phenotypic selection.Methods Enzymol.154, 367-382 (1987).
  5. R. S. Haun et al., Rapid, reliable ligation-independent cloning of PCR products using modified plasmid vectors.BioTechniques13, 515-518 (1992).
  6. K. Wang et al., A simple method using T4 DNA polymerase to clone polymerase chain reaction products.BioTechniques.17, 236-238 (1994).
  7. H-M. Eun, Enzymology Primer for Recombinant DNA Technology (Academic Press, Inc., 1996).

Protocols and Product Manuals

pdf   T4 DNA Polymerase MSDS
pdf   EP0061 - Product Information