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  • RecAf

    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).


    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Rec A Reaction Buffer10X

    Advantages and Features

    Features


    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)

    Properties and Usage

    Notes

    1. ATPγS which is required for triple helix formation is not supplied.

    References

    1. West, S.C. (1992). Ann. Rev. Biochem. 61, 603-640.
    2. Radding, C.M. (1991). J. Biol. Chem. 266
    3. Wasserman, S.A. and Cozzarelli, N.R. (1985). Proc. Natl. Acad. Sci. USA. 82, 1079-1083.
    4. Shortle, D. et al. (1980). Proc. Natl. Acad. Sci. USA. 77, 5375-5379.
    5. Honigberg, S.M. et al. (1986). Proc. Natl. Acad. Sci. USA. 83, 9586-9590.
    6. Rigas, B. et al. (1986). Proc. Natl. Acad. Sci. USA. 83, 9591-9595.
    7. Ferrin, L.J. and Camerini-Otero, R.D. (1991). Science. 254, 1494-1497.
    8. Koob, M. et al. (1992). Nucleic Acids Res. 20, 5831-5836.
    9. Koob, M. et al. (1992). In R. Wu(Ed.), Methods in Enzymology. 216, 321-329. San Diego: Academic Press.
    10. Zhumabayeva, B. et al. (1990). Biotechniques. 27, 834-845.
    11. Zhumabayeva, B. et al. (2001). Biotechniques. 30, 512-520.
    12. Pace, C.N. et al. (1995). Protein Sci. 4, 2411-2423.

    FAQs

    1. What buffer should be used with RecAf Protein?
    2. Is magnesium required for triple strand formation using RecAf Protein?
    3. Is ATP gamma-S required for triple strand formation using RecAf Protein?
    4. Can the RecAf Protein be heat inactivated?
    5. Is the structure of the target DNA important when using RecAf Protein?
    6. How much RecAf Protein should be added to the single stranded DNA to coat it?
    7. What length single stranded oligo should be used with RecAf Protein?

    Tech Tips

    It’s N-term 6XHis tagged RecA with moderately stronger ssDNA binding ability compared to wt RecA. A possible reason might be the increased pI due to the attachment of the 6XHis-tag.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

    Usage Guidelines & Tips

    Quality Control

    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.

    Quality Control Assays

    The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
    • Endonuclease Activity (Nicking):
      The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
    • Exonuclease Activity (Radioactivity Release):
      The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
    • Molecular Weight Determination (Mass Spectrometry) :
      The molecular weight of the product is determined using mass spectrometry.
    • Non-Specific DNase Activity (16 hour):
      The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.
    • Protein Concentration (A280):
      The protein concentration is determined by A280 spectroscopy using the Pace method (Pace, C.N. et al. (1995) Protein Sci., 4, 2411–2423).
    • RNase Activity (2 Hour Digestion):
      The product is tested in a reaction containing a RNA substrate.  After incubation for 2 hour there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.

    Safety Data Sheet

    The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

    Datacards

    The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.