Attention Golden Gate Assembly users: BsaI-HFv2 (NEB #R3733) has been optimized for Golden Gate Assembly*. BsaI-HFv2 also works well for any protocol requiring DNA cutting by BsaI. This is the recommended enzyme for any purpose requiring digestion at the recognition sequence ...5′-GGTCTC(N1)/(N5)-3′...
*The requirements for digestion of DNA during Golden Gate Assembly are more demanding than what is encountered in traditional DNA cloning. For Golden Gate, the enzyme must function well in a buffer that might not be the same buffer as that normally provided for the restriction enzyme, and maintain activity for an extended time at elevated temperatures in a dynamic cutting/re-ligating reaction with competition for substrate binding between the endonuclease and ligase. Extensive testing has demonstrated superior performance of BsaI-HFv2 compared to both BsaI (NEB #R0535) and BsaI-HF (NEB #R3535) in this challenging Golden Gate assembly context where restriction enzyme efficiency and fidelity are critically important.
High-Fidelity (HF®) restriction enzymes have the same specificity as native enzymes, but have been engineered for significantly reduced star activity and performance in a single buffer (CutSmart® Buffer). All HF-restriction enzymes come with Gel Loading Dye, Purple (6X). Enjoy the enhanced performance and added value of our engineered enzymes at the same price as the native enzyme:
- Engineered for improved performance
- Used in Golden Gate Assembly
- Type IIS restriction enzymes recognize asymmetric DNA sequences and cleave outside of their recognition sequence
- 100% activity in CutSmart Buffer
- Time-Saver™ qualified for digestion in 5-15 minutes
- Reduced star activity
- Supplied with 1 vial of Gel Loading Dye, Purple (6X)
Restriction Enzymes in Golden Gate Assembly
Golden Gate Assembly Workflow
Reduce Star Activity with High-Fidelity Restriction Enzymes
Time-Saver Protocol for Restriction Enzyme Digests
NEB TV Ep. 15 – Applications of Restriction Enzymes
CutSmart Restriction Enzyme Buffer
Restriction Enzyme Digest Protocol: Cutting Close to DNA End
Restriction Enzyme Digestion Problem: DNA Smear on Agarose Gel
Why is My Restriction Enzyme Not Cutting DNA?
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.
NEB extensively performs quality controls on all standard and high-fidelity (HF) restriction enzymes. Examples of nuclease contamination studies for some of our HF restriction enzymes are shown below.
Product SourceAn E. coli strain that carries the cloned and modified BsaI gene from Bacillus stearothermophilus 6-55 (Z. Chen)
The following reagents are supplied with this product:
Store at (°C) Concentration Gel Loading Dye, Purple (6X) 25 6 X CutSmart® Buffer -20 10 X
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