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  • T4 Gene 32 Protein

    Description

    T4 Gene 32 Protein is a single-stranded DNA (ssDNA) binding protein required for bacteriophage T4 replication and repair (1-2). It cooperatively binds to and stablizes transiently formed regions of ssDNA and plays an important structural role during T4 phage replication (3). It also has been used extensively to stabilize and mark regions of ssDNA for electron microscopic examination of intracellular DNA structures (4-5). Recently, it has been shown to improve restriction enzyme digestion (6), improve the yield and efficiency of reverse transcription (RT) reactions during RT-PCR (7-9), enhance T4 DNA polymerase activity (10-11), as well as increase the yield of PCR products (12).

    Highlights

    • Stabilize and mark ssDNA for electron microscopy
    • Improve yield and efficiency of RT during RT-PCR
    • Isolated from a recombinant source

    Product Source

    An E. coli strain carrying a plasmid that overexpresses the gene 32 protein of T4 phage

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBuffer 4-2010X

    Advantages and Features

    Applications

    • Stabilization and marking of ssDNA structures (4-5)
    • Increase yield and processivity of RT during RT-PCR (7-9)
    • Increase yield and specificity of PCR products from soil samples (10-12)

    Properties and Usage

    Reaction Conditions

    1X NEBuffer 4
    Incubate at 37°C

    1X NEBuffer 4:
    50 mM Potassium Acetate
    20 mM Tris-acetate
    10 mM Magnesium Acetate
    1 mM DTT
    pH 7.9 @ 25°C

    Storage Temperature

    -20°C

    Storage Conditions

    10 mM Tris-HCl
    300 mM NaCl
    1 mM DTT
    0.1 mM EDTA
    50% Glycerol
    pH 7.5 @ 25°C

    Molecular Weight

    Theoretical: 33506 daltons

    Quality Control

    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.
    • 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.
    • Phosphatase activity (FAM labeled oligo):
      The product is tested in a reaction containing a fluorescent internal labeled single stranded oligonucleotide with a 5' phosphate. The percent removal of the 5' phosphate is determined by capillary 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).
    • Protein Purity (SDS-PAGE):
      The physical purity is assessed by comparing contaminating protein bands in a concentrated sample to the protein of interest band in a sample of known dilution. The purity is determined by SDS-PAGE.
    • RNase Activity (1 Hour Digestion):
      The product is tested in a reaction containing a RNA substrate.  After incubation for 1 hour there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.
    • RNase Activity (Extended Digestion):
      The product is tested in a reaction containing a RNA substrate. After incubation for 16 hours greater than 90% of the substrate RNA remains intact as determined by gel electrophoresis.
    • Single Stranded DNA Binding (FAM labeled oligo):
      The product is tested for its ability to produce a mobility shift when incubated using standard reaction conditions with a single stranded FAM labeled oligo.
    • Single Stranded DNase Activity (FAM Labeled Oligo):
      The product is tested in a reaction containing a fluorescent internal labeled single stranded oligonucleotide. The percent degradation is determined by capillary electrophoresis.
    • Single-Stranded DNase Activity (Radioactivity Release):
      The product is tested in reaction containing radiolabeled single-stranded DNA. After incubation the exonuclease activity is determined by the % release of radioactive nucleotides.

    References

    1. Alberts, B. and Frey, L. (1970). Nature. 227, 1313-1318.
    2. Kornberg, A. and Baker, T. DNA Replication. (2nd ed.), 328-332.
    3. Bittner, M. et al. (1979). J. Biol. Chem. 254, 9565-9572.
    4. Delius, H. et al. (1972). J. Mol. Biol.. 67, 341-350.
    5. Wu, M. and Davidson, N. (1975). Proc. Natl. Acad. Sci. USA. 72, 4506.
    6. Dombroski, D.F. and Morgan, A.R. (1985). J. Biol. Chem. 260, 415-447.
    7. Villalva, C. et al. (2001). Biotechniques. 31, 81-86.
    8. Baugh, L.R. et al. (2001). Nucl. Acids Res. 29, e29.
    9. Jeffries, D. and Farquharson, C. (2002). Anal Biochem. 308, 192-194.
    10. Huberman, J.A. and Kornberg, A. (1971). J. Mol. Biol. 62, 39-52.
    11. Topal, M.D. and Sinha, N.K. (1983). J. Biol. Chem. 258, 12274-12279.
    12. Schwartz, K. et al. (1990). Nucl. Acids Res. 18, 1079.

    Supporting Documents

    Material Safety Datasheets

    The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

    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.