Description
Recognition site and hydrolysis position:
GGTCTCN | GGTCTCN↑ |
CCAGAG(N)5 | CCAGAG(N)5↓ |
Source: Bacillus stearothermophilus 31
Unit definition:
One unit of the enzyme is the amount required to hydrolyze 1 μg of T7 DNA in 1 hour at 55°C in a total reaction volume of 50 μl.
Assayed on:
T7 DNA
Optimal SE-buffer: SE-buffer O + BSA
Enzyme activity (%):
B | G | O | W | Y | ROSE |
25 | 75 | 100 | 75 | 25 | 40 |
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.
Ligations:
After 5-fold overdigestion with enzyme more than 90% of DNA fragments can be ligated. Of these 80% can be recut.
Non-specific hydrolisis:
No nonspecific activity was detected after incubation of 1 μg of T7 DNA with 10 u.a. of enzyme for 16 hours at 55°C.
Reagents Supplied with Enzyme:
Methylation sensitivity: Blocked by overlapping Dcm-methylation (CmCWGG): GAGACCWGG
Not blocked by methylation GGTCT(5mC)
Notes: To obtain 100% activity, BSA should be added to the 1x reaction mix to a final concentration of 100 μg/ml.
Do not use BSA for long incubation.
References:
Shinkarenko, N.M., Lebedeva, N.A., Dedkov, V.S., Abdurashitov, M.A., Degtyarev, S.K. Unpublished observations (1999).