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DNA Polymerase I, E.coli 

#EP0041 Supplied with: 10X Reaction Buffer	500 u (10 u/µl) 1 ml
#EP0042 Supplied with: 10X Reaction Buffer	2500 u (10 u/µl) 5x1 ml
Related Documents (in pdf, ~40-60 KB): Certificate of Analysis: #EP0041, #EP0042 MSDS (English) MSDS (English-USA) MSDS (German)

 

Features

• Incorporates modified nucleotides (e.g. biotin-, digoxigenin-, aminoallyl-, fluorescently-labeled nucleotides).
• Active in Fermentas buffers for restriction enzymes, PCR and RT.

Description
The 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.

Source
E.coli cells with a cloned polA gene.

Molecular Weight
103 kDa monomer.

Applications

• DNA labeling by nick-translation in conjunction with DNase I (1-3), see Protocol for DNA Labeling by Nick-translation.
• Second-strand synthesis of cDNA in conjunction with RNase H, see Protocol for Second Strand cDNA Synthesis (4).

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, 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).

Storage Buffer
The enzyme is supplied in: 25 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 1 mM DTT and 50% (v/v) glycerol.

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

Inhibition and Inactivation

• Inhibitors: metal chelators, PP_i, P_i (at high concentrations) (5).
• Inactivated by heating at 75°C for 10 min or by the addition of EDTA.

Related Products

dNTP Set dNTP Mix, 10 mM each Modified Nucleotides (molecular biology grade): Aminoallyl-dUTP  Biotin-11-dUTP Fluorescein-12-dUTP dm6ATP dm4CTP dm5CTP M-MuLV Reverse Transcriptase RevertAid™ M-MuLV Reverse Transcriptase RevertAid™ H Minus M-MuLV Reverse Transcriptase RNase H, E.coli DNase I, RNase-free Pyrophosphatase, Inorganic (from yeast) 0.5 M EDTA, pH 8.0 Water, nuclease-free

References

1. Ausubel, F.M., et al., Current Protocols in Molecular Biology, vol. 1, John Wiley & Sons, Inc., Brooklyn, New York, 3.5.3-3.5.6., 1994-2005.

2. Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor laboratory, Cold Spring Harbor, N. Y., 2001.
3. Yu, H., et al., Cyanine dye dUTP analogs for enzymatic labeling of DNA probes, Nucleic Acids Res., 22, 3226-3232, 1994.
4. Gubler, U., Hoffmann, B.J., A simple and very efficient method for generating cDNA libraries, Gene, 25, 263-269, 1983.
5. Eun, H-M., Enzymology Primer for Recombinant DNA Technology, Academic Press, INC, 1996.

Updated kovo 18, 2008 09:02