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Recognition Site:
isoschizomers | compatible ends | single letter code
Source:
Bacillus stearothermophilus B674 (Z. Chen)
Reagents Supplied:
NEBuffer 4 (10X)
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 50% | | NEBuffer 2: | | 100% | | NEBuffer 3: | | 75% | | NEBuffer 4: | | 100% |
When using a buffer other than the optimal (supplied) NEBuffer, it may be necessary to add more enzyme to achieve complete digestion.
Methylation Sensitivity:
| dam methylation: Blocked by overlapping | | dcm methylation: Not sensitive | | CpG methylation: Blocked by some combinations of overlapping | |
More information about: Methylation Sensitivity |
Activity at 37°C:
20%
Heat Inactivation:
80°C for 20 minutes
Survival in a Reaction:
(+) Intermediate activity. Suitable for extended digestion, but < 8 hours.
More information about: Extended Digests with Restriction Enzymes
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 4
Incubate at
60°C.
1X NEBuffer 4:
20 mM Tris-acetate
50 mM potassium acetate
10 mM Magnesium Acetate
1 mM Dithiothreitol
pH 7.9 @ 25°C
Unit Definition:
One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA in 1 hour at 60°C in a total reaction volume of 50 µl.
Concentration:
10,000 units/ml
Unit Assay Substrate:
λ DNA (dam-)
Storage Conditions:
10 mM Tris-HCl
300 mM NaCl
1 mM Dithiothreitol
0.1 mM EDTA
500 µg/ml BSA
50% Glycerol
pH 7.4 @ 25°C
Storage Temperature:
-20°C
Diluent Compatibility:
Diluent B
Notes
General notes:- Conditions of low ionic strength, high enzyme concentration, glycerol concentration > 5%, or pH > 8.0 may result in star activity.
FAQs
- The NEB catalog has historically stated that activity in NEBuffer 4 is less than 100% yet this enzyme is now supplied with NEBuffer 4. What have you changed?
- Has the conversion to NEBuffer 4 altered any of the properties of the restriction enzyme?
- If I have an old tube of enzyme, what NEBuffer should I use?
- Will the new enzyme work in the originally supplied NEBuffer?
- Why is NEB switching this restriction enzyme to NEBuffer 4?
- What is Star Activity and how can it be avoided?
- Do degenerate recognition sites need to be palindromic?
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Ligation and Re-cutting:
After a 10-fold overdigestion with BsaBI, > 95%
of the DNA fragments can be ligated with T4 DNA Ligase (at a 5' termini concentration of 1-2 μM)
at 16ºC. Of these ligated fragments, > 95% can be recut with BsaBI.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 50 units of BsaBI incubated for 16 hours at 60ºC
resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis. However, fragments produced by noncanonical cleavage due to star activity may be observed with 10 units of enzyme in similar conditions.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 100 units of BsaBI with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 60ºC
released < 0.1% of the total radioactivity.
Reagents Sold Separately
NEBuffer 4
Companion Products
dam-/dcm- Competent E. coli
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New
England Biolabs, Inc. is an ISO 9001 and ISO
14001 Certified Company.
NEB certifies that it is a small business in accordance with the US Small Business Administration and 13 CFR 121.201 |
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