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M0355L |
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1,000 μg
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2 mg/ml |
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M0355S |
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200 μg
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2 mg/ml |
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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:
RecA is expressed as a N-terminal 6X His tagged recombinant protein from a plasmid in E. coli strain ER3010 which encodes a full-length 353 amino acids wild type E. coli RecA protein.
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)
Properties & Usage
Heat Inactivation:
65°C for 20 minutes
Molecular Weight:
Theoretical: 38,796 daltons
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:
Protein concentration is determined by the Pace method (12) using extinction coefficient of 21,555 for recA.
Concentration:
2 mg/ml
Storage Conditions:
10 mM Tris-HCl
1 mM DTT
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.
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:
Each lot is tested for its ability to form a stable triple helix and is visually determined to be > 99% pure on an SDS-polyacrylamide gel.
Exonuclease Activity:
A 50 μl reaction in RecA Reaction Buffer containing 1 µg of a mixture of single and double-stranded [3H] E. coli DNA and 10 µg of RecA incubated for 4 hours at 37°C releases < 0.1% of the total radioactivity.
Endonuclease Activity:
A 50 μl reaction in RecA Reaction Buffer containing 1 µg of supercoiled ΦX174 RF I DNA and 10 µg of RecA incubated for 4 hours at 37°C results in < 10% conversion to the nicked form as determined by agarose gel electrophoresis.
Non-specific Nuclease Assay:
A 50 µl reaction in RecA Reaction Buffer containing 1 µg λ DNA and 10 µg of RecA incubated for 16 hours at 37°C results in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis.
RNase Assay:
A 50 µl reaction in RecA Reaction Buffer containing 40 ng of labeled RNA and 10 µg of RecA is incubated at 37°C. After incubation for 16 hours, >90% of the substrate RNA remains intact as determined by agarose gel electrophoresis.
RecA Functional Assay:
The plasmid pUC19 contains 5 HpyCH4 IV sites. A 60 mer was designed with complementarity to the region centered around the HpyCH4 IV 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 Sss I supplemented with 160 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 HpyCH4 IV were added followed by digestion at 37°C for 20 minutes. > 95% 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.
- Pace, C.N. et al. (1995) Protein Sci, 4, 2411-2423.
- 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) Nucleic Acids Res, 20, 5831-5836.
- Koob, M. et al. (1992) In R. Wu (Eds.), Methods in Enzymology, 216, pp. 321-329. San Diego: Academic Press.
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