Description:

Tth RecA is a RecA homolog isolated from Thermus thermophilus. It has a ssDNA-dependent ATPase activity at an optimal temperature between 65 to 75oC.

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:

Applications:

Visualization of DNA structures for electron microscopy (2)
Site-directed mutagenesis through D-loop (3,4)
Screening of DNA libraries using RecA-probe filaments (5,6)
Targeted cleavage of DNA (7)
Improvement of PCR specificity and yield (8)

Storage Conditions:

Storage Buffer:
10 mM Tris-HCl
100 mM KCl
0.1 mM EDTA
1 mM DTT
0.1% Triton X-100
50% Glycerol
pH7.5 @ 25oC

Storage Temperature:
-20oC

Quality Control:

Quality Assurance Statement:
Tth RecA is purified free of contaminating endonucleases and exonucleases. Each lot is tested for single-strand, DNA-dependent ATPase activity and is visually determined to be > 95% pure on an SDS-polyacrylamide gel.

Exonuclease Activity:
Incubation of 20 μg Tth RecA for 4 hours at 37°C in 50 μl reaction buffer containing 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate and 1 mM dithiothreitol, pH 7.9 @ 25°C, with 1 μg of a mixture of single and double-stranded [³H] E. coli DNA (200,000 cpm/μg) released < 0.05% of the total radioactivity.

Endonuclease Assay:
Incubation of 10 μg Tth RecA for 4 hours at 37°C in 50 μl reaction buffer containing 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate and 1 mM dithiothreitol, pH 7.9 @ 25°C, with 1 μg ΦX174 RF I DNA gave < 5% conversion to RF II.

Nuclease Activity:
Incubation of 20 μg Tth RecA for 16 hours at 37°C in 50 μl of reaction buffer containing 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate and 1 mM dithiothreitol, pH 7.9 @ 25°C, with 1 μg λ DNA yielded a clear and sharp band on an agarose gel.

References:

1. Radding, C. M. (1991) J Biol Chem 266, 5355-8.
2. Wasserman, S. A. & Cozzarelli, N. R. (1985) Proc Natl Acad Sci U S A 82, 1079-83.
   
3. Biet, E., Maurisse, R., Dutreix, M. & Sun, J. (2001) Biochemistry 40, 1779-86.
   
4. Shortle, D., Koshland, D., Weinstock, G. M. & Botstein, D. (1980) Proc Natl Acad Sci U S A 77, 5375-9.
   
5. Rigas, B., Welcher, A. A., Ward, D. C. & Weissman, S. M. (1986) Proc Natl Acad Sci U S A 83, 9591-5.
   
6. Honigberg, S. M., Rao, B. J. & Radding, C. M. (1986) Proc Natl Acad Sci U S A 83, 9586-90.
   
7. Koob, M., Burkiewicz, A., Kur, J. & Szybalski, W. (1992) Nucleic Acids Res 20, 5831-6.
   
8. Shigemori, Y., Mikawa, T., Shibata, T. & Oishi, M. (2005) Nucleic Acids Res 33, e126.
9. Tth RecAH0200STth RecAH0200S