- Nick translation of DNA
- Second strand cDNA synthesis
- Supplied with 10X Reaction Buffer
Product SourceAn E. coli strain that carries an overexpressed copy of the polA gene.
The following reagents are supplied with this product:
|Store at (°C)||Concentration|
Advantages and Features
- Nick translation of DNA to obtain probes with a high specific activity (2)
- Second strand synthesis of cDNA (3,4)
Properties and Usage
Unit DefinitionOne unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid insoluble material in 30 minutes at 37°C.
1X NEBuffer 2
Incubate at 37°C
1X NEBuffer 2:
50 mM NaCl
10 mM Tris-HCl
10 mM MgCl2
1 mM DTT
pH 7.9 @ 25°C
25 mM Tris-HCl
1 mM DTT
0.1 mM EDTA
pH 7.4 @ 25°C
Heat Inactivation75°C for 20 min
Molecular WeightTheoretical: 103000 daltons
5' - 3' ExonucleaseYes
3' - 5' ExonucleaseYes
Unit Assay Conditions1X NEBuffer 2, 33 μM dNTPs including [3H]-dTTP and 70 μg/ml denatured herring sperm DNA.
Quality Control AssaysThe following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
- Endonuclease Activity (Nicking):
The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
- DNase I is not included with this enzyme and must be added for nick translation reactions.
- DNA Polymerase I (E.coli) is active in all four NEBuffers when supplemented with dNTPs (not included).
- Lehman, I.R. (1981). In P.D. Boyer(Ed.), The Enzymes. 14A, 16-38. San Diego: Academic Press.
- Meinkoth, J. and Wahl, G.M (1987). S.L. Berger and A.R. Kimmel(Ed.), Methods Enzymol.. 152, 91-94. San Diego: Academic Press.
- Gubler, U. and Hoffmann, B.J. (1983). Gene. 25, 263-269.
- D'Alessio, J.M. and Gerard, G.F. (1988). Nucl. Acids Res.. 16, 1999-2014.
- Kunkel, T.A., Loeb, L.A. and Goodman, M.F. (1984). J. Biol. Chem. . 259, 1539-1545.
- Can DNA Polymerase I (E. coli) be used in other NEBuffers?
- Can DNA Polymerase I (E. coli) be used to blunt DNA?
- Can DNA Polymerase I (E. coli) be used to fill in 3' overhangs?
- Why are there artifacts in my blots using the nick translated probe?
- Are NEB DNA Polymerases supplied with dNTPs?
- Can DNA Polymerase I (E. coli) be used to remove 5' overhangs?
- Can DNA Polymerase I (E. coli) be heat inactivated?
- Can DNA Polymerase I (E. coli) be used in nick translation protocols?
- Is nick translation the best way to make a labeled probe?