|Source||Streptococcus milleri S|
|Assayed on||T7 DNA (SspI-digest), plasmid DNA|
|Description||Turbo Smi I is used for short time (10 min) DNA digestion in SE-Buffer O+ .
- Fast DNA analysis
- Fast preparation of vectors for cloning
- Double digestion
|Reaction buffer||SE-buffer O+ (50 mM Tris-HCl (pH 7.6 at 25°C); 10 mM MgCl2; 100 mM NaCl; 1 mM DTT; 100 μg/ml BSA.)|
|Storage conditions||10 mM Tris-HCl (pH 7.5); 250 mM NaCl; 0.1 mM EDTA; 7 mM 2-mercaptoethanol; 100 μg/ml BSA; 50% glycerol. Store at -20°C.|
|Ligation||After digestion with 1 μl of Turbo Smi I, approximately 95% 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.
|Reagents Supplied with Enzyme||
10 X SE-buffer O+|
|Methylation sensitivity||not tested|
|Inactivation 20 minutes under||65oC|
1 μl of Turbo Smi I cuts 1 μg of DNA in 1x SE-Buffer O+ in 10 min (assayed on T7 DNA/SspI 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 O+ - 2 μl
DNA - 0.2-1 μg
Nuclease-free water - to 20 μl
+ 1 μl of Turbo Smi I
Mix by pipette tip carefully.
Incubate at 37°C for 10 min.
|References:||Dedkov, V.S., Bondar, T.S., Shevchenco, A.V., Degtyarev, S.Kh. Mol. Gen. Mikrobiol. Virusol. 1: 23-27 (2000).|