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  • SacI

    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.

    SacI has a High Fidelity (HF) Version available
    cloned at neb recombinant timesaver 5min incubation temp heat inactivation blue white
    Sac-I-cutsite_1
    Catalog #SizeConcentrationPriceQtyAdd to Cart
    R0156S2,000 units20,000 units/ml$55.00Add to Cart
    R0156L10,000 units20,000 units/ml$220.00Add to Cart
    R0156M10,000 units100,000 units/ml$220.00Add to Cart
      
    Categories:
    Restriction Endonucleases: S,
    Time-Saver™ Qualified Restriction Enzymes
    Applications:
    Restriction Enzyme Digestion

    Description

    SacI has a High Fidelity version SacI-HF® (NEB #R3156).

    High Fidelity (HF®) Restriction Enzymes have 100% activity in CutSmart™ Buffer; single-buffer simplicity means more straightforward and streamlined sample processing. HF enzymes also exhibit dramatically reduced star activity. HF enzymes are all Time-Saver™ qualified and can therefore cut substrate DNA in 5-15 minutes with the flexibility to digest overnight without degradation to DNA. Engineered with performance in mind, HF restriction enzymes are fully active under a broader range of conditions, minimizing off-target products, while offering flexibility in experimental design.

    Product Source

    An E. coli strain that carries the SacI gene from Streptomyces achromogenes (ATCC 12767).

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    NEBuffer 1.1-2010X

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA (HindIII digest) in 1 hour at 37°C in a total reaction volume of 50 µl.

    Reaction Conditions

    1X NEBuffer 1.1
    Incubate at 37°C

    1X NEBuffer 1.1:
    10 mM Bis-Tris-Propane-HCl
    10 mM MgCl2
    100 μg/ml BSA
    pH 7 @ 25°C

    Activity in NEBuffers

    NEBuffer 1.1: 100%
    NEBuffer 2.1: 50%
    NEBuffer 3.1: 10%
    CutSmart™ Buffer: 100%

    Diluent Compatibility

    Storage Temperature

    -20°C

    Storage Conditions

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

    Heat Inactivation

    65°C for 20 min

    Methylation Sensitivity

    dam methylation: Not Sensitive
    dcm methylation: Not Sensitive
    CpG Methylation: Not Sensitive

    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.
    • Blue-White Screening (Terminal Integrity):
      A sample of DNA vector linearized with a 10-fold excess of a restriction endonuclease, religated and transformed into an E. coli strain expressing the LacZ beta fragment gene results in less than 1% white colonies.
    • 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.
    • 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. SacI is inhibited by salt concentrations > 10 mM. Mini-prep DNA containing residual salt is resistant to cleavage. A 70% alcohol wash or dialysis can be used to remove the salt.
    2. SacI is sensitive to cytosine methylation at GAGmCTC but not GAGCTmC and insensitive to adenine methylation at GmAGCTC.

    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. Why isn't SacI cutting?
    2. How can the efficiency of SacI be increased?
    3. Is SacI affected by methylation?
    4. What is the molecular weight of SacI?
    5. What is the activity of SacI at 25°C?
    6. What effect does BSA have on the performance of NEB’s restriction enzymes when included in the new buffers?
    7. Do I have to set-up digests with Time-Saver™ qualified enzymes for 5-15 minutes? Can I digest longer?
    8. How can I access the old NEBuffer Activity Chart?
    9. How can I access the old Double Digest Finder?

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