Bar I

Supplied in:

20mM KH2PO4 (pH 7.4); 100mM KCl; 0.1mM EDTA; 200 µg/ml BSA,7mM  2-mercaptoethanol; 50% glycerol.

Reaction Conditions:

1X SEBuffer 2K

Incubate at 37°C.

Warranty period for the enzyme storage at -20˚C is two years from the date of the last assay indicated on the enzyme vial.

1X SEBuffer 2K (pH 7.6 @ 25ºC)

10mM Tris-HCl, 200mM KCl, 10mM MgCl2, 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 37°C in a total reaction volume of  50 µl.

Quality Control Assays

Ligation:

After 3-fold overdigestion with Bar I, ~90% of T7 DNA fragments can be ligated with T4 DNA Ligase and ~95% of these can be recut.

16-Hour Incubation:

A 50 ml reaction containing 1µg of T7 DNA and 4 units of enzyme incubated for 16 hours resulted in the same pattern of DNA bands as a reaction incubated for 1 hour.

Oligonucleotide Assay:

No detectable degradation of a single- and double-stranded oligonucleotide was observed after incubation with 2 units of enzyme for 3 hours.

Enzyme Properties

Activity in SEBuffers:

SEBuffer B             0%

SEBuffer G             0-10%

SEBuffer O           25-50%

SEBuffer W          50-75%

SEBuffer Y           10-25%

SEBuffer ROSE   40%

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:

10X SEBuffer 2K