Description:

The extreme thermostability of ET RecA proves to be ideal for 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:
ET 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 ET 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 ET 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 ET 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. ET RecAH0210SET RecAH0210S