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.
- Engineered for improved performance
- Used in Golden Gate Assembly; maintains high activity levels in T4 DNA Ligase Buffer often used in this application
- Type IIS restriction enzymes recognize asymmetric DNA sequences and cleave outside of their recognition sequence
- 100% activity in rCutSmart Buffer
- Time-Saver™ qualified for digestion in 5-15 minutes
- Reduced star activity
- NEB has developed convenient kits (using BsmBI-v2 and BsaI-HFv2) for performing Golden Gate Assembly.
- Cut Site: GGTCTC(1/5)
"I really, really love BsaI-HFv2. We are back to getting phenomenal GGA results again with very little background."
- -M.C, Principal Investigator, Davidson College, NC
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.
1X rCutSmart™ Buffer
Incubate at 37°C
1X rCutSmart™ Buffer
50 mM Potassium acetate
20 mM Tris-acetate
10 mM Magnesium acetate
100 µg/ml Recombinant Albumin
(pH 7.9 @ 25°C)
Activity in NEBuffers
NEBuffer™ r1.1: 100%
NEBuffer™ r2.1: 100%
NEBuffer™ r3.1: 100%
rCutSmart™ Buffer: 100%
10 mM Tris-HCl
200 mM NaCl
1 mM DTT
0.1 mM EDTA
200 µg/ml BSA
pH 7.4 @ 25°C
80°C for 20 min
dam methylation: Not Sensitive
dcm methylation: Impaired by Some Combinations of Overlapping
CpG Methylation: Blocked by Some Combinations of Overlapping