Klenow Fragment, exo-
#EP0421
Supplied with:
10X Reaction Buffer300 u (5 u/µl)
1 ml#EP0422
Supplied with:
10X Reaction Buffer1500 u (5 u/µl)
5x1 mlRelated Documents (in pdf, ~40-60 KB):
Certificate of Analysis: #EP0421, #EP0422
MSDS (English)
MSDS (English-USA)
MSDS (German)
Features
- Incorporates modified nucleotides (e.g., Cy3-, Cy5-, fluorescein-, rhodamine-, aminoallyl-, biotin-labeled nucleotides).
- Active in Fermentas buffers for restriction enzymes, PCR and RT.
Description
The Klenow Fragment, exo-, is the Large Fragment of DNA Polymerase I, E.coli. 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 (1).Source
E.coli cells with a cloned DNA fragment of the mutated polA gene.Molecular Weight
68 kDa monomer.Applications
- Random-primed DNA labeling (2-4), see Protocol for Random-primed DNA Labeling.
- Labeling of double-stranded DNA recessed 3'-termini, see Protocol for Labeling Recessed 3'-termini of Double-stranded DNA.
- Strand displacement amplification (SDA) (5).
- DNA sequencing by the Sanger method (6).
Quality Control
The absence of endodeoxyribonucleaes and exodeoxyribonucleases confirmed by appropriate quality tests. Functionally tested in random-primed DNA labeling.Concentration
5 u/µlDefinition 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 8.0 at 25°C), 50 mM MgCl2, 10 mM DTT.Inhibition and Inactivation
- Inhibitors: metal chelators, PPi, Pi (at high concentrations) (7).
- Inactivated by heating at 75°C for 10 min or by the addition of EDTA.
Note
The Klenow Fragment, exo-, is not recommended for fill-in reactions prior to DNA ligation, since it frequently adds one or more extra nucleotides to the 3’-terminus of a blunt-ended DNA substrate in a non-template directed fashion (8).
Related Products
- dNTP Mixes
- dNTP Set
- Modified Nucleotides (molecular biology grade):
Aminoallyl-dUTP
Biotin-11-dUTP
Fluorescein-12-dUTP
dm6ATP
dm4CTP
dm5CTP- DecaLabel™ DNA Labeling Kit
- Biotin DecaLabel™ DNA Labeling Kit
- Pyrophosphatase, Inorganic (from yeast)
- 0.5 M EDTA, pH 8.0
- Water, nuclease-free
Derbyshire, V., et al., Genetic and crystallographic studies of the 3’,5’-exonucleolytic site of DNA polymerase I, Science, 240, 199-201, 1988.
- Feinberg, A.P., Vogelstein, B., A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity, Anal. Biochem., 132, 6-13, 1983.
- Feinberg, A.P., Vogelstein, B., Addendum to: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity, Anal. Biochem., 137, 266-267, 1984.
- Yu, H., et al., Cyanine dye dUTP analogs for enzymatic labeling of DNA probes, Nucleic Acids Res., 22, 3226-3232, 1994.
- Walker, G.T., Empirical aspects of strand displacement amplification, PCR Methods Appl., 3, 1-6, 1993.
- Sanger, F., et al., DNA sequencing with chain-terminating inhibitors, Proc. Natl. Acad. Sci. USA, 74, 5463-5467, 1977.
- Eun, H-M., Enzymology Primer for Recombinant DNA Technology, Academic Press, Inc., 1996.
- Clark, J.M., et al., Novel blunt-end addition reactions catalyzed by DNA polymerase I of Escherichia coli, J. Mol. Biol., 198, 123-127, 1987.
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Updated kovo 18, 2008 09:42
