Klenow Fragment
#EP0051
Supplied with:
10X Reaction Buffer300 u (10 u/µl)
1 ml#EP0052
Supplied with:
10X Reaction Buffer1500 u (10 u/µl)
5x1 ml#EP0054
Supplied with:
10X Reaction BufferLC,300 u (2 u/µl)
1 mlRelated Documents (in pdf, ~40-60 KB):
Certificate of Analysis: #EP0051, #EP0052, #EP0054
MSDS (English)
MSDS (English-USA)
MSDS (German)
Features
- Incorporates modified nucleotides (e.g., Cy3-, Cy5-, aminoallyl-, biotin-, digoxigenin- and fluorescently-labeled nucleotides).
- Active in Fermentas buffers for restriction enzymes, PCR and RT.
Description
The Klenow Fragment is the Large Fragment of DNA Polymerase I, E.coli. It exhibits 5’=>3’ polymerase activity and 3’=>5’ exonuclease (proofreading) activity, but lacks 5’=>3’ exonuclease activity of DNA polymerase I.Source
E.coli cells with a cloned fragment of the polA gene.Molecular Weight
68 kDa monomer.Applications
Filling-in or labeling recessed 3'-termini of double-stranded DNA (1), see Protocol for Labeling Recessed 3'-termini of Double-stranded DNA and Protocol for Fill-in Recessed 3'-termini of Double-stranded DNA.
Random-primed DNA labeling (2-4).
DNA sequencing by the Sanger method (5).
Site-specific mutagenesis of DNA with synthetic oligonucleotides (6).
Second strand synthesis of cDNA (7).
Quality Control
The absence of endodeoxyribonucleases confirmed by appropriate quality test. Functionally tested for filling-in of recessed 3’-termini of DNA and for DNA labeling.Concentration
10 u/µl
2 u/µl, LCDefinition 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) (8).
- Inactivated by heating at 75°C for 10 min or by the addition of EDTA.
Related Products
- dNTP Mixes
- dNTP Set
- 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
- Pyrophosphatase, Inorganic (from yeast)
- 0.5 M EDTA, pH 8.0
- Water, nuclease-free
Ausubel, F.M., et al., Current Protocols in Molecular Biology, vol. 1, John Wiley & Sons, Inc., Brooklyn, New York, 3.5.7-3.5.10, 1994-2005.
- Feinberg, A.P., Vogelstein, B., A technique for radiolabeling DNA restriction endonucleases 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.
- Sanger, F., et al., DNA sequencing with chain-terminating inhibitors, Proc. Natl. Acad. Sci. USA, 74, 5463-5467, 1977.
- Wallace, R.B., et al., Directed deletion of a yeast transfer RNA intervening sequence, Science, 209, 1396-1400, 1980.
- Rougeon, F., et al., Insertion of rabbit beta-globin gene sequence into an E.coli plasmid, Nucleic Acids Res., 2, 2365-2378, 1975.
- Eun, H-M., Enzymology Primer for Recombinant DNA Technology, Academic Press, Inc., 1996.
| Home | Search | Contacts | Order | Catalog | Support |
Updated kovo 18, 2008 09:41
