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Restriction Endonucleases >
Restriction Endonucleases >
SfiI |
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Recognition Site:
isoschizomers | compatible ends | single letter code
Source:
A E. coli strain that carries the SfiI gene from Streptomyces fimbriatus (ATCC 15051).
Reagents Supplied:
NEBuffer 4 (10X)
BSA (100X)
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 0% | | NEBuffer 2: | | 100% | | NEBuffer 3: | | 10% | | 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: Impaired by overlapping | | CpG methylation: Blocked by some combinations of overlapping |
Activity at 37°C:
10%
Heat Inactivation:
No
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 4
Supplemented with 100 μg/ml Bovine Serum Albumin
Incubate at
50°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 Adeno-2 DNA in 1 hour at 50°C in a total reaction volume of 50 µl.
Concentration:
20,000 units/ml
Unit Assay Substrate:
Adenovirus-2 DNA
Storage Conditions:
20 mM Tris-HCl
300 mM NaCl
5 mM 2-Mercaptoethanol
0.1 mM EDTA
200 µg/ml BSA
50% Glycerol
0.15% Triton X-100
pH 7.4 @ 25°C
Storage Temperature:
-20°C
Diluent Compatibility:
Diluent C
Notes
General notes:- SfiI requires two copies of its recognition sequence for cleavage to occur. The two sites can be on either the same or different DNA molecules. Wertzell L.M. et al., (1995) J. Mol. Biol. 248:581-595.
FAQs
- What is the minimum distance needed between two SfiI sites? Can they follow each other directly?
- Can I achieve digestion of a plasmid with a single SfiI site?
- The NEB catalog has historically stated that activity in NEBuffer4 is less than 100% yet this enzyme is now supplied with NEBuffer4. What have you changed?
- Has the conversion to NEBuffer4 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?
- Do degenerate recognition sites need to be palindromic?
- How can this enzyme be inactivated?
- Why isn't the enzyme cutting completely?
- Can I add an oligo to my reaction so that two sites are available for efficient cutting?
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 SfiI, > 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 SfiI.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 200 units of SfiI incubated for 16 hours at 50ºC
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 200 units of SfiI with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 50ºC
released < 0% of the total radioactivity.
Endonuclease Activity:
Incubation of a 50 μl reaction containing 100 units of SfiI with 1 μg of
ΦX174 RF I DNA for 4 hours at 50ºC resulted
in < 10% conversion to RFII as determined by agarose gel electrophoresis.
SfiI crystals (Lydia Dorner and Ira Schildkraut, New England Biolabs)
Reagents Sold Separately
NEBuffer 4
BSA
Companion Products
dam-/dcm- Competent E. coli
Legal
Patents:
New England Biolabs, Inc.: U.S. Patent No. 5,637,476
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