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Klenow Fragment

FastDigest buffer for 100% activity Low concentration available 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

Klenow Fragment is the Large Fragment of DNA Polymerase exhibiting 5'→3' polymerase, 3'→5' exonuclease (proofreading) activities, but lacking 5'→3' exonuclease activity.
  
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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)
  
Storage Condition-20 C
HazardousNo
10X Reaction Buffer500mM 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).
InactivationInactivated by heating at 75°C for 10min or by addition of EDTA.
InhibitionInhibitors: metal chelators, PPi, Pi (at high concentrations) (see Reference 8).
Molecular Weight68kDa 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.
SourceE. coli cells with a cloned fragment of the polA gene.
Storage BufferThe enzyme is supplied in: 25mM Tris-HCl (pH7.5), 0.1mM EDTA, 1mM DTT, and 50%(v/v) glycerol.

References

  1. F. M. Ausubel et al., Current Protocols in Molecular Biology, Vol. 1 (John Wiley & Sons, New. York, NY, 1995) pp. 1.4.1-1.4.5, 1.5.5-2.
  2. A. P. Feinberg, B. Vogelstein, A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity.Anal. Biochem.132, 6-13 (1983).
  3. A. P. Feinberg, B. Vogelstein, Addendum to: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity.Anal. Biochem.137, 266-267 (1984).
  4. H. Yu et al., Cyanine dye dUTP analogs for enzymatic labeling of DNA probes.Nucleic Acids Res.22, 3226-3232 (1994).
  5. F. Sanger et al., DNA sequencing with chain-terminating inhibitors.Proc. Natl. Acad. Sci. USA.74, 5463-5467 (1977).
  6. R. B. Wallace et al., Directed deletion of a yeast transfer RNA intervening sequence.Science.209, 1396-1400 (1980).
  7. F. Rougeon et al., Insertion of rabbit beta-globin gene sequence into an E.coli plasmid.Nucleic Acids Res.2, 2365-2378 (1975).
  8. H-M. Eun, Enzymology Primer for Recombinant DNA Technology (Academic Press, Inc., San Diego, California, 1996).

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