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FAQs for RecA Protocols for RecA FAQs for DNA Modifying Enzymes and Cloning Technical Reference for DNA Modifying Enzymes and Cloning Enzyme Finder NEBcutter NEBuffer Chart Double Digest Finder Isoschizomers DNA Sequences and Maps REBASE

Home > Products > DNA Modifying Enzymes and Cloning > Single-stranded DNA Binding Proteins > RecA RecA Catalog # Size Concentration Price Qty   M0249L 1,000 μg 2 mg/ml $244.00 M0249S 200 μg 2 mg/ml $61.00 Prices are in US dollars and valid only for US orders. Download: MSDS PDF Isolated from a recombinant source Visualization of DNA structures with electron microscopy D-loop mutagenesis Screening of DNA libraries > 95% pure as visualized on an SDS-polyacrylamide gel Description: E. coli RecA is necessary for genetic recombination, reactions involving DNA repair and UV-induced mutagenesis. RecA promotes the autodigestion of the lexA repressor, umuD protein and lambda repressor. Cleavage of LexA derepresses more than 20 genes (1). In vitro studies indicate that in the presence of ATP, RecA promotes the strand exchange of single-strand DNA fragments with homologous duplex DNA. The reaction has three distinct steps: (i) RecA polymerizes on the single-strand DNA, (ii) the nucleoprotein filament binds the duplex DNA and searches for a homologous region, (iii) the strands are exchanged (2). Source: An E. coli strain ER2502 that carries an overexpressed RecA gene from E. coli. Applications: Visualization of DNA structures with electron microscopy (3) D-loop mutagenesis (4) Screening libraries using RecA-coated probes (5,6) Cleavage of DNA at any single predetermined site (7,8,9) RecA mediated affinity capture for full length cDNA cloning (10, 11) Reagents Supplied: RecA Reaction Buffer (10X) Reaction & Storage Conditions Reaction Conditions: 1X RecA Reaction Buffer Incubate at 37°C. 1X RecA Reaction Buffer: 70 mM Tris-HCl 10 mM MgCl2 5 mM Dithiothreitol pH 7.6 @ 25°C Unit Definition: Sold by mass of pure protein as determined by OD280 (A280 = 0.516 at 1 mg/ml, 1 cm) Concentration: 2 mg/ml Storage Conditions: 20 mM Tris-HCl 1 mM Dithiothreitol 0.1 mM EDTA 50% Glycerol pH 7.5 @ 25°C Storage Temperature: -20°C Notes General notes: ATPγS which is required for triple helix formation is not supplied. FAQs What buffer should be used with RecA Protein? Is magnesium required for triple strand formation using RecA Protein? Is ATP gamma-S required for triple strand formation using RecA Protein? Can the RecA Protein be heat inactivated? Is the structure of the target DNA important when using RecA Protein? How much RecA Protein should be added to the single stranded DNA to coat it? What length single stranded oligo should be used with RecA Protein? 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-strand, DNA-dependent ATPase activity and is visually determined to be > 95% pure on an SDS-polyacrylamide gel. Exonuclease Activity: Incubation of 10 µg RecA for 4 hours at 30°C in 50 µl 1X reaction buffer containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM dithiothreitol and 1 µg sonicated [3H] DNA (105 cpm/µg) released 0.015% acid soluble counts. Endonuclease Assay: Incubation of 10 µg RecA for 4 hours at 30°C in 50 µl reaction buffer containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM dithiothreitol and 1 µg ΦX174 RF I DNA gave < 5% conversion to RF II. Nuclease Activity: Incubation of 6 µg RecA for 16 hours at 30°C in 50 µl of reaction buffer containing 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM dithiothreitol and 1 µg λ DNA yielded a clear and sharp band on an agarose gel. Ribonuclease Activity: Incubation of 6 µg RecA with 2 µg MS2 phage RNA for 1 hour at 30°C in 50 µl 1X T4 Polynucleotide Kinase Buffer followed by agarose gel electrophoresis gave no change in banding. RecA Functional Assay: The plasmid pUC19 contains 5 HpyCH4IV sites. A 60 mer was designed with complementarity to the region centered around the HpyCH4IV site at position 374. A reaction containing 1 μg pUC19, 0.18 μg 60 mer, 0.3 mM ATP γ-S, 4 μg RecA, in 40 μl 1X RecA Reaction Buffer was incubated at 37°C for 10 minutes to form a stable triple helix. The unprotected sites were methylated using 8 units of SssI supplemented with160 μM SAM for 10 minutes at 37°C. The reaction was stopped and the triple helix was disrupted by incubation at 65°C for 15 minutes. The reaction was cooled and 10 units of HpyCH4IV were added followed by digestion at 37°C for 20 minutes. > 90% of the product is single cut pUC19. References West, S.C. (1992) Ann. Rev. Biochem., 61, 603-640. Zhumabayeva, B. et al. (1990) Biotechniques, 27, 834-845. Zhumabayeva, B. et al. (2001) Biotechniques, 30, 512-520. Radding, C.M. (1991) J. Biol. Chem., 266, 5355-5358. Wasserman, S.A. and Cozzarelli, N.R. (1985) Proc. Natl. Acad. Sci. USA, 82, 1079-1083. Shortle, D. et al. (1980) Proc. Natl. Acad. Sci. USA, 77, 5375-5379. Honigberg, S.M. et al. (1986) Proc. Natl. Acad. Sci. USA, 83, 9586-9590. Rigas, B. et al. (1986) Proc. Natl. Acad. Sci. USA, 83, 9591-9595. Ferrin, L.J. and Camerini-Otero, R.D. (1991) Science, 254, 1494-1497. Koob, M. et al. (1992) Nucl. Acids Res., 20, 5831-5836. Koob, M. (1992) R. Wu (Eds.), Methods in Enzymology, 216, pp. 321-329. San Diego: Academic Press.