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  • BsaI-HF®

    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.

    High Fidelity Enzymes

    Product Update: A free vial of Gel Loading Dye, Purple (6x)  is now included with all HF restriction enzymes.

    cloned at neb recombinant engineered timesaver 5min incubation temp heat inactivation cpg dcm
    R3535
    Catalog #SizeConcentrationPriceQtyAdd to Cart
    R3535S1,000 units20,000 units/ml$64.00Add to Cart
    R3535L5,000 units20,000 units/ml$258.00Add to Cart
      
    Categories:
    High-Fidelity (HF®) Restriction Endonucleases,
    Restriction Endonucleases: B,
    Time-Saver™ Qualified Restriction Enzymes
    Applications:
    Golden Gate Assembly,
    Restriction Enzyme Digestion

    Description

      
    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 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 cloned and modified BsaI gene from Bacillus stearothermophilus 6-55 (Z. Chen)

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    CutSmart® Buffer-2010X
    Gel Loading Dye, Purple (6X)256X

    Properties and Usage

    Unit Definition

    One unit is defined as the amount of enzyme required to digest 1 µg of pXba DNA 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: 50%
    NEBuffer 2.1: 100%
    NEBuffer 3.1: 25%
    CutSmart™ Buffer: 100%

    Diluent Compatibility

    Storage Temperature

    -20°C

    Storage Conditions

    10 mM Tris-HCl
    200 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: Blocked by Overlapping
    CpG Methylation: Blocked by Some Combinations of Overlapping

    FAQs

    1. What does HF® refer to following the name of a restriction enzyme?
    2. When is star activity a concern?
    3. Why does the HF version of the enzyme have a different recommended buffer than the wild type enzyme?
    4. How does the level of star activity of BsaI-HF compare to BsaI?
    5. What is the specific activity of BsaI-HF?
    6. Is there a difference in cutting close to the ends between BsaI-HF and BsaI?
    7. When should I choose the High Fidelity (HF) version of the enzyme?
    8. How is the improvement in fidelity of HF restriction endonucleases quantitated?
    9. What is the Fidelity Index (FI)?
    10. Can the change in buffer preference of the HF enzyme be advantageous?
    11. Will the HF enzyme produce elevated star activity when used in a buffer other than the one recommended?
    12. What does it mean to be Time-Saver™ qualified?
    13. What effect does BSA have on the performance of NEB’s restriction enzymes when included in the new buffers?
    14. Do I have to set-up digests with Time-Saver™ qualified enzymes for 5-15 minutes? Can I digest longer?
    15. How can I access the old NEBuffer Activity Chart?
    16. How can I access the old Double Digest Finder?
    17. 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?
    18. Which restriction enzymes are used in Golden Gate Assembly?
    19. Which restriction enzymes are used in GoldenBraid Assembly?
    20. Is Gel Loading Dye, Purple (6X) or Gel Loading Dye, Purple (6X), no SDS compatible with other DNA binding dyes such as SYBR® and GelRed™ during gel electrophoresis?
    21. Can Gel Loading Dye, Purple 6X (B7024) be stored in cold temperatures?

    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 Tools

    Usage Guidelines & Tips

    Troubleshooting Guides

    Interactive Tools

    Citations

    • Abil Z, Denard CA, Zhao H (2014). Modular assembly of designer PUF proteins for specific post-transcriptional regulation of endogenous RNA J Biol Eng. 8(1), 7. PubMedID: 24581042, DOI: 10.1186/1754-1611-8-7
    • Sakuma T, Nishikawa A, Kume S, Chayama K, Yamamoto T (2014). Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system Sci Rep. 4, 5400. PubMedID: 24954249, DOI: 10.1038/srep05400
    • Binder A, Lambert J, Morbitzer R, Popp C, Ott T, Lahaye T, Parniske M (2014). A Modular Plasmid Assembly Kit for Multigene Expression, Gene Silencing and Silencing Rescue in Plants PLoS One. 9(2), e88218. PubMedID: 24551083, DOI: 10.1371/journal.pone.0088218
    • Alejandro Sarrion-Perdigones, Marta Vazquez-Vilar, Jorge Palací, Bas Castelijns, Javier Forment, Peio Ziarsolo, José Blanca, Antonio Granell, Diego Orzaez (2013). GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology Plant Physiology. 162(3), 1618-31. PubMedID: 23669743

    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.
    • 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.