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Restriction Endonucleases >
Restriction Endonucleases >
BbsI |
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Recognition Site:
isoschizomers | compatible ends | single letter code
Source:
Bacillus laterosporus (C. Story)
Reagents Supplied:
NEBuffer 2
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 100% | | NEBuffer 2: | | 100% | | NEBuffer 3: | | 25% | | NEBuffer 4: | | 75% |
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: Not sensitive | | dcm methylation: Not sensitive | | CpG methylation: Not sensitive |
Heat Inactivation:
65°C for 20 minutes
Survival in a Reaction:
Minimum units to digest 1 µg of substrate DNA in 16 hours: 0.25 unit(s)
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 2
Incubate at
37°C.
1X NEBuffer 2:
10 mM Tris-HCl
50 mM NaCl
10 mM MgCl2
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 37°C in a total reaction volume of 50 µl.
Concentration:
5,000 units/ml
Unit Assay Substrate:
λ DNA
Storage Conditions:
10 mM Tris-HCl
300 mM KCl
1 mM Dithiothreitol
0.1 mM EDTA
300 µg/ml BSA
50% Glycerol
pH 7.5 @ 25°C
Storage Temperature:
-20°C
For long term storage (>30 days), store at -70°C.
Diluent Compatibility:
Diluent B
Notes
General notes:- BbsI is an isoschizomer of Bbv II.
- -70°C storage is recommended for periods greater than 30 days. Avoid freeze/thaw cycles.
FAQs
- Are storage conditions for BbsI other than -20°C recommended?
- Is activity loss of BbsI seen in 12 months or less?
- Does salt inhibit BbsI cleavage?
- What is the activity of BbsI at 25°C?
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 20-fold overdigestion with BbsI, > 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 BbsI.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of λDNA
and 50 units of BbsI incubated for 16 hours at 37ºC
resulted in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 50 units of BbsI with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (105 cpm/μg) for 4 hours at 37ºC
released < 0.1% of the total radioactivity.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 10 units of BbsI with 1 μg of
pUC19 DNA for 4 hours at 37ºC resulted
in < 10% conversion to RFII as determined by agarose gel electrophoresis.
Reagents Sold Separately
NEBuffer 2
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