Klenow Fragment, exo-

FastDigest buffer for 100% activity LO certified 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, exo- is the large fragment of DNA polymerase I. Exhibits 5'→3' polymerase activity, but lacks the 3'→5' and 5'→3' exonuclease activities.
  
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Thermo Scientific Klenow Fragment, exo-, is the large fragment of DNA polymerase I . It exhibits 5'→3' polymerase activity, but lacks the 3'→5' and 5'→3' exonuclease activities of DNA Polymerase I. The 3'→5' exonuclease activity of the enzyme is eliminated by mutations in the 3'→5'-exonuclease active site ( see Reference1).

Highlights

  • Lacks 3'→5' exonuclease activity
  • Incorporates modified nucleotides (e.g., Cy3-, Cy5-, fluorescein-, rhodamine-, aminoallyl-, biotin-labeled nucleotides)
  • Active in restriction enzyme, PCR, and RT buffers

Applications

  • Random-primed DNA labeling ( see ​ References 2-4)
  • Labeling by fill-in 5'-overhangs of dsDNA
  • Strand displacement amplification (SDA) ( see ​ Reference 5)
  • DNA sequencing by the Sanger method ( see ​ Reference 6)

Note

Klenow Fragment, exo- is not recommended for DNA blunting reactions prior to DNA ligation since it frequently adds one or more extra nucleotides to the 3'-terminus of blunt-end DNA substrates in a non-template directed fashion ( see ​ Reference8).

  
10X Reaction Buffer500 mM Tris-HCl (pH8.0 at 25°C), 50mM MgCl 2 , 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 30min 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 MgCl 2 , 1mM 2-mercaptoethanol, 0.033mM dATP, 0.033mM dTTP, 0.4MBq/mL [ 3 H]-dTTP, and 62.5µg/mL poly(dA-dT)·poly(dA-dT).
HazardousNo
InactivationInactivated by heating at 75°C for 10min or by the addition of EDTA.
InhibitionInhibitors: metal chelators, PP i , P i (at high concentrations) ( see Reference 7).
Molecular Weight68kDa monomer.
Quality Control
  • The absence of endo- and exodeoxyribonucleases confirmed by appropriate quality tests.
  • Functionally tested in random-primed DNA labeling.
SourceE. coli cells with a cloned DNA fragment of the mutated polA gene.
Storage BufferThe enzyme is supplied in:
25mM Tris-HCl (pH7.5), 0.1mM EDTA, 1mM DTT, and 50%(v/v) glycerol.
Storage Condition-20 C

References

  1. V. Derbyshire et al. , Genetic and crystallographic studies of the 3',5'-exonucleolytic site of DNA polymerase I.Science.240 , 199-201 (1988).

  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. G. T. Walker, Empirical aspects of strand displacement amplification.PCR Methods Appl.3 , 1-6 (1993).

  6. F. Sanger et al. , DNA sequencing with chain-terminating inhibitors.Proc. Natl. Acad. Sci. USA.74 , 5463-5467 (1977).

  7. H-M. Eun, Enzymology Primer for Recombinant DNA Technology (Academic Press, Inc., San Diego, California, 1996).

  8. J. M. Clark et al. , Novel blunt-end addition reactions catalyzed by DNA polymerase I of Escherichia coli . J. Mol. Biol.198 , 123-127 (1987).

Citations