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Restriction Endonucleases >
Restriction Endonucleases >
SfcI |
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Recognition Site:
isoschizomers | compatible ends | single letter code
Source:
An E. coli strain that carries the cloned SfcI gene from Streptococcus faecium (A. Meloni)
Reagents Supplied:
NEBuffer 4
BSA
Enzyme Properties
Activity in NEBuffers:
| NEBuffer 1: | | 75% | | NEBuffer 2: | | 50% | | 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: 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: 1.00 unit(s)
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 4
Supplemented with 100 μg/ml Bovine Serum Albumin
Incubate at
37°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 37°C in a total reaction volume of 50 µl.
Concentration:
10,000 units/ml
Unit Assay Substrate:
λ DNA
Storage Conditions:
10 mM Tris-HCl
300 mM KCl
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
Usage notes:- SfcI concentrations of < 1 unit/µg DNA in a reaction are not recommended.
- SfcI is active for one hour in a reaction at 37°C, after one hour no further digestion takes place. The stability of SfcI is greatly enhanced by incubations at 25°C. Although the enzyme is only 25% as active at this temperature, we recommend an incubation temperature of 25°C for 4 - 16 hours when digesting supercoiled plasmids. At 25°C, 0.1 unit will digest 1µg of substrate DNA in 16 hours.
FAQs
- 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 2-fold overdigestion with SfcI, > 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 SfcI.
16-Hour Incubation:
A 50 μl reaction containing 1 μg of DNA
and 50 units of SfcI 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 SfcI with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 37ºC
released < 1% of the total radioactivity.
Reagents Sold Separately
NEBuffer 4
BSA
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