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T7 DNA Polymerase

#EP0081 Supplied with: 10X Reaction Buffer	300 u (10 u/µl) 0.4 ml
Related Documents (in pdf, ~40-60 KB): Certificate of Analysis: #EP0081 MSDS (English) MSDS (English-USA) MSDS (German)

 

Features

• Removes residual genomic DNA from preparations of covalently closed circular DNA.
• Active in Fermentas buffers for restriction enzymes.

Description
The 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.

Molecular Weight
The 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).

Source
Two E.coli strains, one with the cloned gene 5 of bacteriophage T7, and the other with the cloned trxA gene of E.coli.

Applications

• Purification of covalently closed circular DNA.
• Primer extension reactions on long templates (1).
• DNA 3'-end labeling (1).
• Strand extensions in site-directed mutagenesis (2).
• Fill-in of recessed 3'-termini of double-stranded DNA.
• Second strand synthesis of cDNA (4).
• In situ detection of DNA fragmentation associated with apoptosis (5).

Quality Control
The absence of endodeoxyribonucleases confirmed by appropriate quality test.

Concentration
10 u/µl

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.

Storage Buffer
The enzyme is supplied in: 20 mM potassium phosphate (pH 7.4), 1 mM DTT, 0.1 mM EDTA and 50% (v/v) glycerol.

10X Reaction Buffer
400 mM Tris-HCl (pH 7.5 at 25°C), 100 mM MgCl₂, 10 mM DTT.

Inhibition and Inactivation

• Inhibitors: metal chelators, modification reagents (acetic anhydride, N-ethylmaleimide inactivate the 3’=>5’ exonuclease activity but not the polymerase activity) (6).
• Inactivated by heating at 75°C for 10 min.

Note

• T7 DNA Polymerase has 3'=>5' exonuclease activity approximately 1000-fold greater than that of Klenow fragment (1).
• Assays at 37°C require only short incubation times (3). 

Related Products

dNTP Mix, 10 mM each dNTP Set M-MuLV Reverse Transcriptase RevertAid™ M-MuLV Reverse Transcriptase RevertAid™ M-MuLV Reverse Transcriptase Nb.Bpu10I T4 DNA Ligase 0.5 M EDTA, pH 8.0 Water, nuclease-free

References

1. Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, Third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.

2. Bebenek, K., Kunkel, T.A., The use of native T7 DNA polymerase for site-directed mutagenesis, Nucleic Acids Res., 17, 5408, 1989.
3. Bodescot, M., Brison, O., T7 DNA polymerase requires unusual reaction conditions for blunt-ending activity, Anal. Biochemistry, 216, 234-235, 1994.
4. Bodescot, M., Brison, O., Efficient second-strand cDNA synthesis using T7 DNA polymerase, DNA and Cell Biology, 13, 977-985, 1994.
5. Wood, K.A., 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.
6. Eun, H-M., Enzymology Primer for Recombinant DNA Technology, Academic Press, Inc., 1996.

Updated vasario 05, 2008 14:37