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DNA Polymerase I

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

DNA Polymerase I is a template-dependent DNA polymerase which catalyzes 5'→3' synthesis of DNA.
  
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Thermo Scientific DNA Polymerase I, a template-dependent DNA polymerase, catalyzes 5'→3' synthesis of DNA. The enzyme also exhibits 3'→5' exonuclease (proofreading) activity, 5'→3' exonuclease activity, and ribonuclease H activity.

Highlights

  • Incorporates modified nucleotides (e.g. biotin-, digoxigenin-, aminoallyl-, fluorescently-labeled nucleotides)
  • Active in multiple buffers, including restriction enzyme, PCR, and RT buffers

Applications

  • DNA labeling by nick-translation in conjunction with DNase (see References 1-3)
  • Second-strand synthesis of cDNA in conjunction with RNase H (see Reference 4)
  
Storage Condition-20 C
HazardousNo
10X Reaction Buffer500 mM Tris-HCl (pH 7.5 at 25°C), 100 mM MgCl2, 10 mM DTT.
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 30 min at 37°C, using poly(dA-dT)·poly(dA-dT) as a template·primer.
  • Enzyme activity is assayed in the following mixture: 67 mM potassium phosphate (pH 7.4), 6.7 mM MgCl2, 1 mM 2-mercaptoethanol, 0.033 mM dATP, 0.033 mM dTTP, 0.4 MBq/mL [3H]-dTTP, and 62.5 µg/mL poly(dA-dT)·poly(dA-dT).
InactivationInactivated by heating at 75°C for 10 min or by addition of EDTA.
InhibitionInhibitors: metal chelators, PPi, Pi (at high concentrations) (see Reference 5).
Molecular Weight103 kDa monomer
Quality ControlThe absence of endodeoxyribonucleases confirmed by appropriate quality test.
SourceE.coli cells with a cloned polA gene.
Storage BufferThe enzyme is supplied in 25 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 1 mM DTT, and 50% (v/v) glycerol.

References

  1. F. M. Ausubel et al., Current Protocols in Molecular Biology, vol. 1 (John Wiley and Sons, Inc., Brooklyn, New York, 1994-2005) pp. 3.5.3-3.5.6.
  2. J. Sambrook, D. W. Russell, Molecular Cloning: A Laboratory Manual, Third Edition (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 2001).
  3. H. Yu et al., Cyanine dye dUTP analogs for enzymatic labeling of DNA probes. Nucleic Acids Res. 22, 3226-3232 (1994).
  4. U. Gubler, B. J. Hoffmann, A simple and very efficient method for generating cDNA libraries. Gene. 25, 263-269 (1983).
  5. H. M. Eun, Enzymology Primer for Recombinant DNA Technology (Academic Press, Inc., San Diego, California, 1996).

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