Source: Bacillus stearothermophilus S
Assayed on: Lambda DNA
Unit definition: One unit of the enzyme is the amount required to hydrolyze 1 μg of Lambda DNA in 1 hour at 55°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
Activity in SEBuffers:
SEBuffer B 50 - 75%
SEBuffer G 100%
SEBuffer O 50 - 75%
SEBuffer W 75 -100%
SEBuffer Y 75 -100%
SEBuffer ROSE 100%
When using a buffer other than the optimal (suppied) SEBuffer, it may be necessary to add more enzyme to achieve complete digestion.
Optimal temperature: 55°C
Storage conditions: 10 mM Tris-HCl (pH 7.5); 50 mM KCl; 0,1 mM EDTA; 7 mM 2-mercaptoethanol; 200 μg/ml BSA; and 50% glycerol;
Store: at -20°C.
Ligation: After 5-fold overdigestion with enzyme 80% of the DNA fragments can be ligated. Of these, 95% can be recut.
Non-specific hydrolisis: No nonspecific activity was detected after incubation of 1 μg of Lambda DNA with 10 u.a. of enzyme for 16 hours at 55°C.
Reagents supplied with Enzyme: 10 X SE-buffer G, BSA
Methylation sensitivity: Not blocked by overlapping dcm-methylation (CmCWGG): GKGCCCWGG
Blocked by GKG(5mC)MC methylation
Inactivation: 20 minutes under 65°C
Notes: 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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