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Polymerases and Amplification >
Mesophilic DNA Polymerases >
DNA Polymerase I (E. coli) |
 | | | | DNA Polymerase I (E. coli) | | | |
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- Nick translation of DNA
- Second strand cDNA synthesis
- Supplied with 10X Reaction Buffer
- Specific Activity: 10,000 units/mg
Description:
DNA Polymerase I is a DNA-dependent DNA polymerase with inherent 3´→ 5´ and 5´→ 3´ exonuclease activities (1). The 5´→ 3´ exonuclease activity removes nucleotides ahead of the growing DNA chain, allowing nick-translation.
Source:
An E. coli strain that carries an overexpressed copy of the polA gene.
Applications:- Nick translation of DNA to obtain probes with a high specific activity (3)
- Second strand synthesis of cDNA (4,5)
Reagents Supplied:
NEBuffer 2 (10X)
Enzyme Properties
Polymerase Properties | Thermophilic Polymerase Characteristics
3´ to 5´ Exonuclease: Yes
5´ to 3´ Exonuclease: Yes
Strand Displacement: No
Error Rate: 9
x10-6 bases
Heat Inactivation:
75°C for 20 minutes
Molecular Weight:
Theoretical: 109,000 daltons
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 2
Supplemented with 33 μM dNTPs (not included)
Incubate at
25°C.
1X NEBuffer 2:
10 mM Tris-HCl
50 mM NaCl
10 mM MgCl2
1 mM Dithiothreitol
pH 7.9 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid-insoluble material in a total reaction volume of 50 μl in 30 minutes at 37°C in 1X EcoPol Reaction Buffer with 33 µM dNTPs including [3H]-dTTP and 70 µg/ml denatured herring sperm DNA.
Unit assay Conditions:
1X NEBuffer 2, 33 μM dNTPs including [3H]-dTTP and 70 μg/ml denatured herring sperm DNA.
Concentration:
10,000 units/ml
Storage Conditions:
25 mM Tris-HCl
1 mM Dithiothreitol
0.1 mM EDTA
50% Glycerol
pH 7.4 @ 25°C
Storage Temperature:
-20°C
Notes
Usage notes:- DNase I is not included with this enzyme and must be added for nick translation reactions.
- DNA Polymerase I is also active in all four NEBuffers when supplemented with dNTPs.
FAQs
- Can DNA Polymerase I be used in other NEBuffers?
- Can DNA Polymerase I be used to blunt DNA?
- Can DNA Polymerase I 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 be used to remove 5' overhangs?
- Can DNA Polymerase I be heat inactivated?
- Can DNA Polymerase I be used in nick translation protocols?
- Is nick translation the best way to make a labeled probe?
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 25 units of DNA Polymerase I (E. coli) with 1 μg of
ΦX174 RF I DNA for 4 hours at 37ºC resulted
in < 10% conversion to RFII as determined by agarose gel electrophoresis.
References
- Lehman, I.R. (1981) P.D. Boyer (Eds.), The Enzymes, 14A, pp. 16-38. San Diego: Academic Press.
- Murray, N.E. and Kelley, W.S. (1979) Molec. Gen. Genet., 175, 77-87.
- Meinkoth, J. and Wahl, G.M (1987) S.L. Berger and A.R. Kimmel (Eds.), Methods Enzymol., 152, pp. 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.
Reagents Sold Separately
NEBuffer 2
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