|Source||An E.coli strain, that carries the cloned gene Sfi I from Streptomyces fimbriatus|
|Assayed on||T7 DNA|
|Unit definition||One unit of the enzyme is the amount required to hydrolyze 1 μg of T7 DNA in 1 hour at 50°C in a total reaction volume of 50 μl.|
|Optimal SE-buffer||G (10 mM Tris-HCl (pH 7.6 at 25°C); 10 mM MgCl2; 50 mM NaCl; 1 mM DTT.) + BSA|
|Enzyme activity (%)|
|75 - 100||100||25 - 50||25 - 50||25 - 50||75|
|Storage conditions||10 mM Tris-HCl (pH 7.5); 200 mM NaCl; 0.1 mM EDTA; 7 mM 2-mercaptoethanol; 200 μg/ml BSA; 50% glycerol. Store at -20°C.|
|Ligation||After 10-fold overdigestion with enzyme more than 70% of the DNA fragments can be ligated and recut. In the presence of 10% PEG ligation is better.|
|Non-specific hydrolisis||No nonspecific activity was detected after incubation of 1 μg of T7 DNA with 20 u.a. of enzyme for 16 hours at 50°C.
|Reagents Supplied with Enzyme||
10 X SE-buffer G, BSA
For E123T and E124T an additional 10X SE-buffer ROSE+ is supplied.
|Methylation sensitivity||Blocked by overlapping Dcm methylation (CmCWGG): GGCCWGGNNGGCC.|
Not blocked by overlapping Dcm methylation (CmCWGG): GGCCNNNNNGGCCWGG.
|Inactivation 20 minutes under||65oC|
|Notes||Incubation at 37°C rusults in 75-100% activity.|
To obtain 100% activity, BSA should be added to the 1 x reaction mix to a final concentration of 100 μg/ml.
Do not use BSA for long incubation.
Turbo Sfi I can be used for short time (10 min) DNA digestion as well as for standard reaction. The reaction can be performed using optimal or universal (ROSE+) Buffer
Turbo DNA Digestion:
-Fast DNA analysis
-Fast preparation of vectors for cloning
1 μl of Turbo Sfi I cuts 1 μg of DNA in 1 x SE-Buffer G with BSA or universal 1 x SE-Buffer ROSE+ in 10 min (see the protocol below). A short time of DNA digestion requires high quality purification of DNA sample. The enzyme may be used in standard conditions of DNA cleavage (E123).
Turbo reaction protocol:
20 μl of the reaction volume:
10 x SE Buffer ROSE+ - 2 μl
DNA - 0,2-1 μg
Nuclease-free water - to 20 μl
+ 1μl of Turbo Sfi I
Incubate at 37°C for 10 min.
|Quality control||Restriction Endonucleases: Quality Control|
|MSDS:||Download MSDS as PDF|
|References:||Qiang, B.-Q., Schildkraut, I. Nucleic Acids Res. 12: 4507-4515 (1984).
Dedkov V.S., Degtyarev S. Kh. The resctriction endonucleases detection in colonies of microorganisms Streptomyces and Nocardia // APPLIED BIOCHEMISTRY and MICROBIOLOGY (Russia) Vol 28, 1992, #. 2, 309- 313