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  • Antarctic Phosphatase

    Description

    Antarctic Phosphatase catalyzes the removal of 5´ phosphate from DNA and RNA. Since phosphatase-treated fragments lack the 5´ phosphoryl termini required by ligases, they cannot self-ligate (1). This property can be used to decrease the vector background in cloning strategies.

    Highlights

    1. Removes 5' phosphates from DNA, RNA, rNTPs and dNTPs
    2. Prevents recircularization of cloning vectors
    3. Heat inactivated in 5 minutes at 70°C
    4. Allows ligation without purifying vector DNA
    5. Isolated from a recombinant source

    Product Source

    An E. coli strain that carries the TAB5 AP gene, originally cloned in plasmid pNI (2), recloned in plasmid pEGTAB7-4.1(3).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    Antarctic Phosphatase Reaction Buffer-2010X

    Advantages and Features

    Applications

    • Removing 5´ phosphates from DNA, RNA, rNTPs and dNTPs
    • Preparation of templates for 5´ end labeling
    • Prevention of recircularization of cloning vectors
    • Removal of dNTPs and pyrophosphate from PCR reactions
    • Dephosphorylation of proteins

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme that will dephosphorylate 1 µg of pUC19 vector DNA cut with HindIII (5´ protruding ends), HincII (blunts ends) or PstI (5´ recessed ends) in 30 minutes at 37°C. Dephosphorylation is defined as > 95% inhibition of recirculation in a self-ligation reaction and is measured by transformation into E. coli.

    Reaction Conditions

    1X Antarctic Phosphatase Reaction Buffer
    Incubate at 37°C

    1X Antarctic Phosphatase Reaction Buffer:
    50 mM Bis-Tris-Propane-HCl
    1 mM MgCl2
    0.1 mM ZnCl2
    pH 6 @ 25°C

    Storage Temperature

    -20°C

    Storage Conditions

    10 mM Tris-HCl
    1 mM MgCl2
    1 mM DTT
    50% Glycerol
    0.01 mM ZnCl2
    pH 7.4 @ 25°C

    Heat Inactivation

    70°C for 5 min

    Molecular Weights

    Apparent: 35 kDa
    Theoretical: 69 kDa

    Unit Assay Conditions

    Vector DNA is dephosphorylated in restriction endonuclease buffer supplemented with Antarctic Phosphatase Reaction Buffer. Ligation is performed with 50 ng of vector using the NEB Quick Ligation Kit (NEB #M2200).

    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.
    • Functional Test (Dephosphorylation):
      The Alkaline Phosphatase is tested in a reaction with linearized DNA.  After incubation the DNA is ligated and transformed into competent cells.  The degree of dephosphorylation is determined by comparison to linearized DNA that is not treated with the alkaline phosphatase.
    • 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 (2 Hour Digestion):
      The product is tested in a reaction containing a RNA substrate.  After incubation for 2 hours there is no detectable degradation of the RNA substrate as determined by gel electrophoresis.

    Notes

    1. Adding 1/10 volume of 10X Antarctic Phosphatase Reaction Buffer will provide the amount of Zn2+ that the enzyme requires for activity.

    References

    1. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, (2nd ed.). 5.72.
    2. Rina, M. et al. (2000). Eur. J. Biochem.. 267, 1230-1238.
    3. Guthrie, E., unpublished results. Unpublished observation

    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.
    1. Which alkaline phosphatase, rSAP, CIP, or Antarctic Phosphatase, works best?
    2. The number of colonies that don't contain an insert seems high, how can I tell if the Antarctic Phosphatase worked?
    3. Can I use the enzyme to de-phosphorylate proteins?
    4. Will the enzyme remove P from ssRNA?
    5. Will the enzyme remove 3’ P from DNA?
    6. Can Antarctic Phosphatase remove the 3' phosphate from DNA?
    7. Will Antarctic Phosphatase work in restriction enzyme NEBuffers?
    8. Does the DNA need to be purified after the restriction digest, prior to Antarctic Phosphatase treatment?
    9. Does the DNA need to be purified after Antarctic Phosphatase treatment?
    10. What is the molecular weight?
    11. Can Antarctic Phosphatase be heat inactivated?
    1. Protocol for Dephosphorylation of 5'-ends of DNA using AnP (M0289)

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