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Restriction Endonucleases >
Restriction Endonucleases >
BsmFI |
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Recognition Site:
isoschizomers | compatible ends | single letter code
Source:
An E. coli strain that carries the cloned BsmFI gene from Bacillus stearothermophilus F (ER2683)
Reagents Supplied:
NEBuffer 4
BSA
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 25% | | NEBuffer 2: | | 50% | | NEBuffer 3: | | 50% | | 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: Not sensitive | | dcm methylation: Blocked by overlapping | | CpG methylation: Blocked by overlapping |
Activity at 37°C:
50%
Heat Inactivation:
80°C for 20 minutes
Survival in a Reaction:
Minimum units to digest 1 µg of substrate DNA in 16 hours: 0.13 unit(s)
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 4
Supplemented with 100 μg/ml Bovine Serum Albumin
Incubate at
65°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 pBR322 DNA in 1 hour at 65°C in a total reaction volume of 50 µl.
Concentration:
2,000 units/ml
Unit Assay Substrate:
pBR322 DNA
Storage Conditions:
10 mM Tris-HCl
50 mM NaCl
1 mM Dithiothreitol
0.1 mM EDTA
200 µg/ml BSA
50% Glycerol
pH 7.4 @ 25°C
Storage Temperature:
-20°C
Diluent Compatibility:
Diluent A
Notes
General notes:- BsmFI is an isoschizomer of FinI.
- Occasionally, BsmFI has been shown to cleave the sequence GGGAC(9/13).
The exact frequency of this occurrence has yet to be determined.
FAQs
- Are there single cleavage sites in any common vectors for BsmFI?
- Is there variability in the cleavage site for BsmFI?
- Is activity loss seen in 12 months or less?
- What is the activity of BsmFI at 37°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 5-fold overdigestion with BsmFI, > 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 BsmFI.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of pBR322 DNA
and 5 units of BsmFI incubated for 16 hours at 65ºC
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 20 units of BsmFI with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 65ºC
released < 0.1% of the total radioactivity.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 10 units of BsmFI 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 4
BSA
Companion Products
dam-/dcm- Competent E. coli
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