| Recognition site | G↑GWCC
CCWG↓G |
| Source | Bacillus megaterium 18 |
| Assayed on | Lambda DNA, plasmid DNA |
| Description | Turbo Bme18 I is used for a short time (15 min) of 1 μg DNA digestion in universal (ROSE) SE-Buffer in 50 μl of reaction mixture.
Reaction Original SibEnzyme (ROSE) Buffer is a specially designed universal reaction buffer for the most Restriction Endonucleases. ROSE Buffer is perfect for DNA cleavage with SE Turbo Restriction Endonucleases and for double digestion.
Applications:
- Fast DNA analysis
- Fast preparation of vectors for cloning
- Double digestion
|
| Reaction buffer | SE-buffer ROSE |
| Optimal temperature | 37oC |
| Storage conditions | 10 mM Tris-HCl (pH 7.5); 100 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 Bme18 I, approximately 90% 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 ROSE |
| Methylation sensitivity | Cleaved of DNA is impaired by overlapping dcm-methylation (CmCWGG):
GGWCCWGG. |
| Inactivation 20 minutes under | 65oC |
| Notes | Enzyme Properties:
1 μl of Turbo Bme18 I cuts 1 μg of DNA in 1x SE-Buffer ROSE in 15 min (assayed on Lambda 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 Bme18 I
Mix by pipette tip carefully.
Incubate at 37°C for 15 min.
|
| References: | Degtyarev S.K., Rechkunova N.I., Grinev A.A., Dedkov V.S. Izv. Sib. Otd. Akad. Nauk SSSR 15: 25-26 (1989).
 Chernukhin V.A, Abdurashitov M.A., Tomilov V.N., Gonchar D.A., Degtyarev S.Kh. Comparative analysis of mouse chromosomal DNA digestion with restriction endonucleases in vitro and in silico // "Ovchinnikov bulletin of biotechnology and physical and chemical biology" V.3, No 4, pp 19-27, 2007
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