Supplied in:

10 mM Tris-HCl (pH 7.5); 50 mM KCl; 0,1 mM EDTA; 200 µg/ml BSA; 7 mM  2-mercaptoethanol; 50% glycerol.

Reaction Conditions:

1XSEBuffer Y+BSA

Incubate at 30°C.

1XSEBuffer Y  (pH 7.9 @ 25ºC)

33 mM Tris-Ac, 66 mM KAc, 10 mM MgAc, 1 mM DTT

Unit Definition:

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

Ligation:

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.

16-Hour Incubation:

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.

Oligonucleotide Assay:

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.

Enzyme Properties

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.

Heat Inactivation:

Yes   (65°C for 20 minutes)

Reagents supplied with enzyme:

10XSEBuffer Y, BSA (10 mg/ml)