T4 DNA Polymerase

Detailní popis

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

• 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 (see Reference1)
• Active in Thermo Scientific restriction enzyme, PCR, RT and T4 DNA Ligase buffers

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)

  5× Reaction Buffer	335mM Tris-HCl (pH8.8 at 25°C), 33mM MgCl2, 5mM DTT, 84mM (NH4)2SO4
  Concentration	5U/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.
  Hazardous	No
  Inactivation	Inactivated by heating at 75°C for 10min.
  Inhibition	Inhibitors: metal chelators, nucleotide analogs 2(p-n-butylanilino)-dATP, N2-(p-n-butylphenyl)-dGTP), SH-blocking compounds (see Reference 7)
  Molecular Weight	104kDa monomer
  Quality Control	The absence of endodeoxyribonucleases confirmed by appropriate quality tests.
  Source	E.coli cells with a cloned gene 43 of bacteriophage T4
  Storage Buffer	The enzyme is supplied in 20mM potassium phosphate (pH7.5), 200mM KCl, 2mM DTT, and 50%(v/v) glycerol.
  Storage Condition	-20 C