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Single-stranded DNA Binding Proteins >
Tth RecA |
 | | | | Tth RecA | | | Developed and produced by BioHelix Corp., a NEB-affiliated company. | |
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Prices are in US dollars and valid only for US orders.
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- Extreme thermostability
- Nucleic acid amplification and sequencing
- Isolated from a recombinant source
Description:
Tth RecA is a RecA homolog isolated from Thermus thermophilus. It has a ssDNA-dependent ATPase activity at an optimal temperature between 65–75°C. The extreme thermostability makes Tth RecA ideal for molecular biology applications that require an elevated temperature condition, such as nucleic acid amplification and sequencing.
Source:
An E. coli strain that carries the cloned RecA gene from Thermus thermophilus.
Applications:- Visualization of DNA structures with electron microscopy (1)
- Site-directed mutagenesis through D-loop (2,3)
- Screening of DNA libraries using RecA-probe filaments (4,5)
- Targeted cleavage of DNA (6)
Reaction & Storage Conditions
Unit Definition:
Sold by mass of pure protein as determined by OD280.
Concentration:
1 mg/ml
Storage Conditions:
10 mM Tris-HCl
100 mM KCl
1 mM DTT
0.1 mM EDTA
50% Glycerol
0.1% Triton X-100
pH 7.5 @ 25°C
Storage Temperature:
-20°C
Notes
Usage notes:- Tth RecA is active in any polymerase buffer. Add 400 ng of the Tth RecA per 50 µl reaction.
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Quality Assurance Statement:
RecA is purified free of contaminating endonucleases and exonucleases. Each lot is tested for single-stranded DNA-dependent ATPase activity and is visually determined to be > 95% pure on an SDS-polyacrylamide gel.
Exonuclease Activity:
Incubation of 20 µg RecA for 4 hours at 65°C in 50 µl reaction buffer containing 50 mM potassium acetate (pH 7.9), 20 mM tris-acetate, 10 mM magnesium acetate and 1 mM dithiothreitol (pH 7.9 at 25°C), with a mixture of single and double-stranded [3H] E. coli DNA (200,000 cpm/µg) released < 0.1% of the total radioactivity.
Endonuclease Activity:
Incubation of 2 µg RecA for 4 hours at 65°C in 50 µl reaction buffer containing 50 mM potassium acetate (pH 7.9), 20 mM tris-acetate, 10 mM magnesium acetate and 1 mM dithiothreitol (pH 7.9 at 25°C), with 1 µg ΦX174 RF I DNA gave < 20% conversion to RF II.
Nuclease Activity:
Incubation of 20 µg RecA for 16 hours at 65°C in 50 µl of reaction buffer containing 50 mM potassium acetate (pH 7.9), 20 mM tris-acetate, 10 mM magnesium acetate and 1 mM dithiothreitol (pH 7.9 at 25°C), with 1 µg λ DNA yielded a clear and sharp band on an agarose gel.
References
- Wasserman, S.A. and Cozzarelli, N.R. (1985) Proc. Natl. Acad. Sci. USA, 82, 1079-1083.
- Biet, E. et al. (2001) Biochemistry, 40, 1779-1786.
- Shortle, D. et al. (1980) Proc. Natl. Acad. Sci. USA, 77, 5375-5379.
- Rigas, B. et al. (1986) Proc. Natl. Acad. Sci. USA, 83, 9591-9595.
- Honigberg, S.M. et al. (1986) Proc. Natl. Acad. Sci. USA, 83, 9586-9590.
- Koob, M. et al. (1992) Nucleic Acids Res., 20, 5831-5836.
- higemori, Y. et al. (2005) Nucleic Acids Res., e126.
Legal
Licenses/Patents/Disclaimers:
Some applications in which this product can be used may be covered by patents issued and applicable in the United States and certain other countries. Because purchase of this product does not include a licenses to perform any patented application, users of this product may be required to obtain a patent license depending upon the particular application in which the product is used.
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