10 mM Tris-HCl (pH 7.5); 50 mM KCl; 0,1 mM EDTA; 200 µg/ml BSA; 7 mM 2-mercaptoethanol; 50% glycerol.
Incubate at 30°C.
1XSEBuffer Y (pH 7.9 @ 25ºC)
33 mM Tris-Ac, 66 mM KAc, 10 mM MgAc, 1 mM DTT
One unit is defined as the amount of enzyme required to digest 1 µg of T7 DNA in 1 hour at 30°C in a total reaction volume of 50 µl.
Concentrated enzymes are diluted to approximately 1000 units/ml with the buffer A [10 mM Tris-HCl (pH 7.6); 50 mM KCl; 0.1 mM EDTA; 1 mM DTT;200 µg/ml BSA; 50% glycerol] before determining their
activity. To obtain 100% activity, BSA should be added to the 1x reaction mix to a final concentration of 100 µg/ml.
Quality Control Assays
After 3-fold overdigestion with Psr I, ~70% of T7 DNA fragments can be ligated with T4 DNA Ligase and ~90% of these can be recut. In the presence of
10% PEG ligation is better.
A 50 µl reaction containing 1 µg of T7 DNA and 1 units of enzyme incubated for 16 hours resulted in the same pattern of DNA bands as a reaction
incubated for 1 hour. No using BSA for long incubation. High enzyme concentration may result in star activity.
No detectable degradation of a single- and double-stranded oligonucleotide was observed after incubation with 1 units of enzyme for 3 hours. Incubation at 37°C results in 20% activity.
Activity in SEBuffers:
SEBuffer B 10-25%
SEBuffer G 10-25%
SEBuffer O 0%
SEBuffer W 0-10%
SEBuffer Y 100%
SEBuffer ROSE 30%
When using a buffer other than the optimal (suppied) SEBuffer, it may be necessary to add more enzyme to achieve complete digestion.
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