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ET RecA

 

Products - Primer Navigator™ Series

Catalog #
Size
Concentration
Price
H0210S
100 µg
1 mg/ml
(Discontinued)

 

Download: MSDS

   

Description:

   


RecA is a DNA binding protein with ssDNA-dependent ATPase activity. It plays vital roles in homologous recombination and DNA repair in bacteria by catalyzing strand exchange at a homologous sequence between single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) (1). In the presence of ATP, the RecA protein first polymerizes on the single-stranded DNA to form a nucleoprotein filament. The nucleoprotein filament binds naked duplex DNA and randomly searches for homology. Once a homologous region is found, the strands are exchanged.

ET RecA (Extreme thermostable RecA) is an E. coli RecA homolog isolated from a hyperthermophilic microorganism. ET RecA has a ssDNA-dependent ATPase activity with an optimal temperature between 75 to 85C.

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:

   


purified from an E. coli strain that overexpresses the recA gene isolated from a hyperthermophilic microorganism.
     
     

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:

   


Concentration:

1mg/ml

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 g 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 g 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 g 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.

 
   
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