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.
(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.
Ligation: After 3-fold overdigestion with Psr I,
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.