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T7 DNA Polymerase

B buffer for 100% activity 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

T7 DNA Polymerase, a template dependent DNA polymerase, catalyzes DNA synthesis in the 5'=>3' direction.
  
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Thermo Scientific T7 DNA Polymerase, a template dependent DNA polymerase, catalyzes DNA synthesis in the 5'=>3' direction. It is a highly processive DNA polymerase allowing continuous synthesis of long stretches of DNA. The enzyme also exhibits a high 3'=>5' exonuclease activity towards single- and double-stranded DNA.

Highlights

  • Strong 3’=>5’ exonuclease activity, approximately 1000-fold greater than Klenow Fragment (see Reference 1)
  • Active in restriction enzyme buffers

Applications

  • Purification of covalently closed circular DNA by removal of residual genomic DNA
  • Primer extension reactions on long templates (see​ Reference 1)
  • DNA 3'-end labeling (see​ Reference 1)
  • Strand extensions in site-directed mutagenesis (see​ Reference 2)
  • Fill-in blunting of 5'-overhang DNA
  • Second strand synthesis of cDNA (see​ Reference 3)
  • In situ detection of DNA fragmentation associated with apoptosis (see​ Reference 4)

Note

Assays at 37°C require only short incubation times (see​ Reference 6).

  
Storage Condition-20 C
HazardousNo
10X Reaction Buffer400 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.
  • Enzyme activity is assayed in the following mixture: 40 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 0.1 mg/ml BSA, 0.33 mM of each dNTP, 0.4 MBq/ml [3H]-dTTP, and 0.5 mM alkali-denatured calf thymus DNA.
InactivationInactivated by heating at 75°C for 10 min.
InhibitionInhibitors: metal chelators, modification reagents (acetic anhydride, N-ethylmaleimide inactivate the 3'=>5' exonuclease activity but not the polymerase activity) (see Reference 5)
Molecular WeightThe T7 DNA Polymerase is composed of two subunits: an 80 kDa polypeptide (the product of gene 5 of bacteriophage T7) and a 12 kDa thioredoxin (from the trxA gene of E. coli).
Quality ControlThe absence of endodeoxyribonucleases is confirmed by the appropriate quality test.
SourceTwo E. coli strains, one with the cloned gene 5 of bacteriophage T7, and the other with the cloned trxA gene of E. coli.
Storage BufferThe enzyme is supplied in:
20 mM potassium phosphate (pH 7.4), 1 mM DTT, 0.1 mM EDTA and 50% (v/v) glycerol.

References

  1. J. Sambrook, D. W. Russell, Molecular Cloning: A Laboratory Manual, Third edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001).
  2. K. Bebenek, T. A. Kunkel, The use of native T7 DNA polymerase for site-directed mutagenesis. Nucleic Acids Res. 17, 5408 (1989).
  3. M. Bodescot, O. Brison, Efficient second-strand cDNA synthesis using T7 DNA polymerase. DNA and Cell Biology. 13, 977-985 (1994).
  4. K.A. Wood et al., In situ labeling of granule cells for apoptosis-associated DNA fragmentation reveals different mechanisms of cell loss in developing cerebellum. Neuron. 11, 621-632 (1993).
  5. H. M. Eun, Enzymology Primer for Recombinant DNA Technology (Academic Press, Inc., 1996).
  6. M. Bodescot, O. Brison, T7 DNA polymerase requires unusual reaction conditions for blunt-ending activity. Anal. Biochemistry. 216, 234-235, (1994).

Protocols and Product Manuals

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