| Recognition site | GGCCNNNN↑NGGCC
CCGGN↓NNNNCCGG |
| Source | An E.coli strain, that carries the cloned gene Sfi I from Streptomyces fimbriatus |
| Assayed on | T7 DNA, plasmid DNA |
| Description | Turbo Sfi I is used for short time (10 min) DNA digestion in universal (ROSE+) SE-Buffer.
Reaction Original SibEnzyme (ROSE+) Buffer is a modified universal ROSE (Reaction Original SibEnzyme) Buffer, specially designed for Restriction Endonucleases that require BSA to obtain 100% activity. The concentration of BSA in 1X ROSE+ Buffer is 100 μg/ml. ROSE+ Buffer is perfect for DNA cleavage with SE Turbo Restriction Endonucleases and for double digestion. Do not use this Buffer for long incubation (16 hours) with the enzymes having star activity..
Applications:
- Fast DNA analysis
- Fast preparation of vectors for cloning
- Double digestion
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| Reaction buffer | SE-buffer ROSE+ |
| Optimal temperature | 50oC |
| 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 digestion with 1 μl of Turbo Sfi I, approximately 70% of the DNA fragments can be ligated with high-activity T4 DNA Ligase and recut. |
| Non-specific hydrolisis | No detectable degradation of a single-stranded and double-stranded oligonucleotide was observed after incubation with 1 μl of restriction endonuclease for 3 hours.
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| Reagents Supplied with Enzyme |
10 X SE-buffer ROSE+ |
| Methylation sensitivity | Blocked by overlapping Dcm methylation (CmCWGG): GGCCWGGNNGGCC.
Not blocked by overlapping Dcm methylation (CmCWGG): GGCCNNNNNGGCCWGG. |
| Inactivation 20 minutes under | 65oC |
| Notes | Enzyme Properties:
1 μl of Turbo Sfi I cuts 1 μg of DNA in 1x SE-Buffer ROSE+ in 10 min (assayed on T7 DNA and plasmid DNA). A short time of DNA digestion requires high quality purification of DNA sample (PCR fragments should be purified after amplification).
Please note that supercoiled plasmid DNA and PCR fragments may have varying rates of cleavage and sometimes need more time to be completely digested.
Standard protocol of Turbo reaction:
20 μl of the reaction volume:
10x SE-Buffer ROSE+ - 2 μl
DNA - 0.2-1 μg
Nuclease-free water - to 20 μl
+ 1 μl of Turbo Sfi I
Mix by pipette tip carefully.
Incubate at 50°C for 10 min.
Incubation at 37°C rusults in 75-100% activity.
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| 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
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