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  • PI-PspI

    The new and current Double Digest Finder and current Activity/Performance Chart for the CutSmart buffer system are available. The previous version of the Double Digest Finder, as well as the previous Version of Activity/Performance Chart that use the former buffer system, are still available for your convenience.
    cloned at neb recombinant unique buffer incubation temp heat inactivation no bsa
    PI-Psp-I-cutsite_1
    Catalog #SizeConcentrationPriceQtyAdd to Cart
    R0695S500 units5,000 units/ml$65.00Add to Cart
    R0695L2,500 units5,000 units/ml$260.00Add to Cart
      
    Categories:
    Homing Endonucleases,
    Restriction Endonucleases: P-R
    Applications:
    Restriction Enzyme Digestion

    Description

    PI-PspI is obtained from a strain of E. coli which expresses the DNA polymerase from the extreme thermophile, Pyrococcus species GB-D (1). PI-PspI is a product of in vivo protein splicing that gives rise to both polymerase and endonuclease from a single polypeptide precursor.

    Product Source

    An E. coli strain that carries the PI-PspI gene from Pyrococcus species (H.W. Jannasch).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBuffer PI-PspI-2010X
    pAKR7 XmnI-linearized Control Plasmid500 μg/ml
    BSA-20100X

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to cleave 1 µg of pAKR7 XmnI-linearized Control Plasmid in 1 hour at 65°C in a total reaction volume of 50 µl.

    Reaction Conditions

    1X NEBuffer PI-PspI
    Supplement with BSA
    Incubate at 65°C

    1X NEBuffer PI-PspI:
    10 mM Tris-HCl
    10 mM MgCl2
    1 mM DTT
    150 mM KCl
    pH 9.2 @ 25°C

    Activity in NEBuffers

    NEBuffer 1.1: 10%
    NEBuffer 2.1: 10%
    NEBuffer 3.1: 10%
    CutSmart™ Buffer: 10%

    Diluent Compatibility

    Storage Temperature

    -20°C

    Storage Conditions

    10 mM Tris-HCl
    300 mM NaCl
    1 mM DTT
    0.1 mM EDTA
    500 μg/ml BSA
    50% Glycerol
    pH 7.4 @ 25°C

    Heat Inactivation

    No

    Activity at Temperature

    @37°C: 5%

    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 (Homing Endonuclease):
      The Homing Endonuclease is incubated with a linearized DNA substrate containing the appropriate cleavage site resulting in the expected sized fragments as determined by agarose gel electrophoresis
    • Ligation and Recutting (Terminal Integrity):
      After an over-digestion of DNA with a restriction endonuclease the percentage of the DNA fragments ligated with T4 DNA ligase and the percentage that can be recut are determined by agarose gel electrophoresis.
    • 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.

    Notes

    1. Homing endonucleases do not have stringently-defined recognition sequences in the way that restriction enzymes do. That is, single base changes do not abolish cleavage but reduce its efficiency to variable extents. The precise boundary of required bases is generally not known. The recognition sequence listed is one site that is known to be recognized and cleaved.
    2. Digests of DNA embedded in agarose should be performed with 1 unit of enzyme per µg of DNA for 3 hours at 50°C.
    3. PI-PspI can remain bound to DNA after cutting and alter migration rate of DNA during electrophoresis. To disrupt binding, add SDS to a final concentration of 0.5% or purify DNA before electrophoresis.
    4. Incubation at 37° results in 5% activity.
    5. Supplied with plasmid DNA. XmnI-linearized  pAkR7 is supplied at 0.5 mg/ml in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA. Cleavage of this 3.7 kb plasmid gives fragments of 2146 and 1532 base pairs.  

    Supporting Documents

    Certificate of Analysis

    The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality control's for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

    Specifications

    The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

    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. How can this enzyme be inactivated?
    2. How can I access the old NEBuffer Activity Chart?
    3. How can I access the old Double Digest Finder?

    Selection Tools

    Usage Guidelines & Tips

    Troubleshooting Guides

    Interactive Tools

    PI-PspI (or PI-SceI or I-CeuI) can remain bound to DNA after cutting and alter migration rate of DNA during electrophoresis.
    To disrupt binding, add SDS to a final concentration of 0.5% or purify DNA before electrophoresis.