| Recognition site | ↑(N)7GAACNNNNNNTAC(N)12↑
↓(N)12CTTGNNNNNNATG(N)7↓ |
| Source | Pseudomonas stutzeri N2 |
| 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 30°C in a total reaction volume of 50 μl. |
| Optimal SE-buffer | Y (33 mM Tris-acetate (pH 7.9 at 25°C); 10 mM magnesium acetate; 66 mM potassium acetate; 1 mM DTT.) + BSA |
| Enzyme activity (%) | | B | G | O | W | Y | R | | 10 - 25 | 10 - 25 | 0 - 10 | 0 - 10 | 100 | 30 |
|
| Optimal temperature | 30oC |
| Storage conditions | 10 mM Tris-HCl (pH 7.5); 50 mM KCl; 0,1 mM EDTA; 7 mM 2-mercaptoethanol; 200 μg/ml BSA, 50% glycerol. Store at -20°C. |
| Ligations | After 2-fold overdigestion with enzyme more than 70% of DNA fragments can be ligated. Of these 80% can be 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 2 u.a. of enzyme for 16 hours at 30°C.
|
| Reagents Supplied with Enzyme |
10 X SE-buffer Y, BSA |
| Methylation sensitivity | not tested |
| Inactivation 20 minutes under | 65oC |
| Notes | High enzyme concentration may result in star activity.
Incubation at 37°C results in 20% 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.
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| MSDS: | Download MSDS as PDF |
| References: | Dedkov, V.S., Kileva, E.V., Abdurashitov, M.A., Gonchar, D.A., Popichenko, D.V., Degtyarev, S.K.
Unpublished observations (2001).
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