Klenow Fragment

Detailní popis

Thermo Scientific Klenow Fragment is the large fragment of DNA polymerase I. It exhibits 5'→3' polymerase activity and 3'→5' exonuclease (proofreading) activity, but lacks 5'→3' exonuclease activity of DNA polymerase I.

Highlights

• Incorporates modified nucleotides (e.g., Cy3-, Cy5-, aminoallyl-, biotin-, digoxigenin- and fluorescently-labeled nucleotides)
• Active in restriction enzyme, PCR, RT, and T4 DNA Ligase buffers

Applications

• DNA blunting by fill-in 5'-overhangs (see Reference 1)
• Random-primed DNA labeling (see References 2-4)
• Labeling by fill-in 5'-overhangs of dsDNA
• DNA sequencing by the Sanger method (see Reference 5)
• Site-specific mutagenesis of DNA with synthetic oligonucleotides (see Reference 6)
• Second strand synthesis of cDNA (see Reference 7) 

  10X Reaction Buffer	500mM Tris-HCl (pH8.0 at 25°C), 50mM MgCl2, 10mM DTT.
  Definition of Activity	Unit One unit of the enzyme catalyzes the incorporation of 10nmol of deoxyribonucleotides into a polynucleotide fraction (adsorbed on DE-81) in 30minutes at 37°C, using poly(dA-dT)·poly(dA-dT) as a  template·primer. Enzyme activity is assayed in the following mixture: 67mM potassium phosphate (pH7.4), 6.7mM MgCl2, 1mM 2-mercaptoethanol, 0.033mM dATP, 0.033mM dTTP, 0.4MBq/mL [3H]-dTTP, and 62.5µg/mL poly(dA-dT)·poly(dA-dT).
  Hazardous	No
  Inactivation	Inactivated by heating at 75°C for 10min or by addition of EDTA.
  Inhibition	Inhibitors: metal chelators, PPi, Pi (at high concentrations) (see Reference 8).
  Molecular Weight	68kDa monomer.
  Quality Control	The absence of endodeoxyribonucleases confirmed by appropriate quality test. Functionally tested for fill in of 5'-overhanging DNA termini and for random primed DNA labeling.
  Source	E. coli cells with a cloned fragment of the polA gene.
  Storage Buffer	The enzyme is supplied in: 25mM Tris-HCl (pH7.5), 0.1mM EDTA, 1mM DTT, and 50%(v/v) glycerol.
  Storage Condition	-20 C