BsaI

Description

free dye




BsaI has a High Fidelity version BsaI-HF™ (NEB #R3535).

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 cloned 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

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: 75%
NEBuffer 2.1: 75%
NEBuffer 3.1: 100%
CutSmart® Buffer: 100%

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

65°C for 20 min

Methylation Sensitivity

dam methylation: Not Sensitive
dcm methylation: Impaired by Some Combinations of Overlapping
CpG Methylation: Blocked by Some Combinations of Overlapping

Notes

  1. Blocked by overlapping dcm methylation. Cleavage of mammalian genomic DNA is blocked by some combinations of overlapping CpG methylation.
  2. Activity at 50°C is 100%.
  3. May exhibit star activity in NEBuffer 1.1.
  4. Based on the stability of the enzyme in the reaction, incubations longer than 1 hr will not result in improved digestion, unless additional enzyme is added. Please refer to Restriction endonuclease survival in a reaction for more information regarding this topic.
  5. Star activity may result from a glycerol concentration of >5%

FAQs

  1. What effect does BSA have on the performance of NEB’s restriction enzymes when included in the new buffers?
  2. How can I access the old NEBuffer Activity Chart?
  3. Which restriction enzymes are used in Golden Gate Assembly?
  4. Which restriction enzymes are used in GoldenBraid Assembly?
  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?

Tech Tips

Restriction Enzymes in Golden Gate Assembly
 

Protocols

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

Selection Charts

Usage Guidelines & Tips

Troubleshooting Guides

Interactive Tools

Citations

  • Feng Y, Zhang S, Huang X (2014). A robust TALENs system for highly efficient mammalian genome editing Sci Rep. 4, 3632. PubMedID: 24407151, DOI: 10.1038/srep03632
  • 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. Further information regarding NEB product quality can be found here.
  • 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.