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    I-SceI

    Description

    I-SceI is an intron-encoded endonuclease. The intron encoding I-SceI is present in the mitochondria of Saccharomyces cerevisiae.

    Product Source

    An E. coli strain that carries the I-SceI mitochondrial gene from Saccharomyces cerevisiae (B. Dujon).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    pGPS2 NotI-linearized Control Plasmid500 μg/ml
    CutSmart® Buffer-2010X

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to cleave 1 µg of pGPS2 NotI-Iinearized Control Plasmid in 1 hour at 37°C in a total reaction volume of 50 µl.

    Reaction Conditions

    1X CutSmart® Buffer
    Incubate at 37°C

    1X CutSmart® Buffer:
    50 mM Potassium Acetate
    20 mM Tris-acetate
    10 mM Magnesium Acetate
    100 μg/ml BSA
    pH 7.9 @ 25°C

    Activity in NEBuffers

    NEBuffer 1.1: 10%
    NEBuffer 2.1: 50%
    NEBuffer 3.1: 25%
    CutSmart® Buffer: 100%

    Diluent Compatibility

    Storage Temperature

    -80°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

    65°C for 20 min

    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. Supplied with plasmid DNA. NotI-linearized pGPS2 is supplied at 0.5 mg/ml in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA. Cleavage of this 2499 bp plasmid with I-SceI gives fragments of 1518 and 981 base pairs.
    3. The enzyme retains full activity after at least 10 free-thaw cycles; the enzyme can be aliquoted and stored at –80C.

    FAQs

    1. My enzyme used to come with another NEBuffer, but CutSmart™ Buffer is now recommended?  Why?
    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. I tested your restriction enzyme on the substrate DNA recommended by NEB, and it appears to be active, however it does not digest my DNA. What could be the reason?
    5. Why is my Restriction Enzyme not cutting DNA?
    6. Why do I see a DNA smear on an agarose gel after a restriction digest?
    7. Why do I see additional DNA bands on my gel after a restriction digest?
    8. How many nucleotides do I have to add adjacent to the RE recognition site in order to get efficient cutting?

    Protocols

    1. Optimizing Restriction Endonuclease Reactions
    2. Double Digest Protocol with Standard Restriction Enzymes

    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.

    Selection Charts

    Usage Guidelines & Tips

    Troubleshooting Guides

    Interactive Tools

    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. Further information regarding NEB product quality can be found here.
    • Endonuclease Activity (Nicking, Buffer):
      The buffer 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.

    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]

    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.