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  • PI-SceI

    This enzyme has transitioned to an improved new buffer system. Visit NEBCutSmart.com for further details.
     
    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.
    recombinant unique buffer incubation temp heat inactivation bsa
    PI-SCEI
    Catalog #SizeConcentrationPriceQtyAdd to Cart
    R0696S250 units5,000 units/ml$65.00Add to Cart
    R0696L1,250 units5,000 units/ml$260.00Add to Cart
      
    Categories:
    Homing Endonucleases,
    Restriction Endonucleases: P-R
    Applications:
    Restriction Enzyme Digestion

    Description

    The intein encoding PI-SceI is present in the VMA ATPase gene Saccharomyces cerevisiae (1,5). The gene has been modified for independent expression in E. coli using a T7 RNA polymerase expression system (2).

    Product Source

    An E. coli strain that carries the VMA1 ATPase gene from Saccharomyces cerevisiae (J. Thorner).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    pBSvdeX XmnI-linearized Control Plasmid500 μg/ml

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to cleave 1 μg of pBSvdeX XmnI-linearized Control Plasmid in 3 hours at 37°C in a total reaction volume of 50 μl.

    Reaction Conditions

    1X NEBuffer PI-SceI
    Supplement with BSA
    Incubate at 37°C

    1X NEBuffer PI-SceI:
    10 mM MgCl2
    1 mM DTT
    10 mM Tris-HCl
    100 mM KCl
    pH 8.6 @ 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
    1 mM DTT
    0.1 mM EDTA
    500 μg/ml BSA
    50% Glycerol
    300 mM NaCl
    pH 7.4 @ 25°C

    Heat Inactivation

    65°C for 20 min

    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.
    • 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. Supplied with plasmid DNA: XmnI-linearized pBSvdeX is supplied 0.5 mg/ml in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA. Cleavage of this 3.7 kb plasmid with PI-SceI gives fragments of 2550 and 1150 base pairs Enzyme Properties
    2. 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.
    3. PI-SceI 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.

    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. Can a double digest be performed with PI-SceI and I-CeuI?
    2. What effect does BSA have on the performance of NEB’s restriction enzymes when included in the new buffers?
    3. How can I access the old NEBuffer Activity Chart?
    4. How can I access the old Double Digest Finder?

    Selection Tools

    Usage Guidelines & Tips

    Troubleshooting Guides

    Interactive Tools

    PI-SceI 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.