10u/ul

Bacillus coagulans VS 29-022

One unit is defined as the amount of B0808-05 required to digest 1ug of control DNA fragments in 1 hour at 37°C in 50ul of assay buffer. The control DNA is pUC19 DNA with inserted BcuI recognition site - Psp1406I fragments.

Supplied as a liquid in 10mM Tris-HCl (pH 7.5 at 25°C), 300mM KCl, 1mM DTT, 0.1mM EDTA, 0.2mg/ml BSA and 50% glycerol.

R1625: Restriction Enzyme Buffer A, 10X: Suplied as a liquid in 33mM Tris-acetate pH 7.9, 10mM magnesium acetate, 66mM potassium acetate, 0.1mg/ml BSA. Incubate at 37°C.

Diluent Buffer: 10mM Tris-HCl (pH 7.4 at 25°C), 100mM KCl, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA and 50% glycerol. For longer periods–the Storage Buffer should be used.

No detectable change in the specific fragmentation pattern is observed after 160-fold overdigestion (10u/ug pUC19-BcuI DNA x 16 hours) with BcuI.

The ligation and recleavage assay was replaced with LO test after validating experiments showed LO test ability to trace nuclease and phosphatase activities with sensitivity that is higher than L/R by a factor of 100.

No detectable degradation of a single-stranded and double-stranded labeled oligonucleotide was observed after incubation with 10 units of BcuI for 4 hours.

The B/W assay was replaced with LO test after validating experiments showed LO test ability to detect nuclease and phosphatase activities with sensitivity that equals to that of B/W test.

A minimum of 0.5units of BcuI is required for complete digestion of 1ug of Ad2 DNA in 16 hours at 37°C.

Dam, Dcm, CpG - Never overlaps, no effect EcoKI, EcoBI - effect not determined

A minimum of 10 units of BcuI is required for complete digestion of 1ug of agarose-embedded Adenovirus-2 DNA in 16 hours.

Eco130I, NheI, XbaI, XmaJI

Ad2: 3 Lambda: 0 PhiX174: 0 M13mp18/19: 0 pBR322: 0 pUC18/19: 0 pUC57: 0 pTZ19R/U: 0

BcuI is not inactivated by incubation at 80°C for 20min.

1. To prepare the digested DNA for electrophoresis: (a) stop the digestion by adding 0.5M EDTA, pH 8.0, to achieve a 20mM final concentration. Mix thoroughly, add an electrophoresis loading dye and load onto gel. 2. To prepare DNA suitable for further enzymatic reactions: (a) extract with phenol/chloroform, precipitate with ethanol or isopropanol, wash the pellet with 75% cold ethanol and air dry; (b) dissolve DNA in either nuclease-free water, TE buffer, or a buffer suitable for further applications; (c) check the DNA concentration in the solution.

No detectable change in the specific fragmentation pattern is observed after 160-fold overdigestion (10u/ug pUC19-BcuI DNA x 16 hours) with BcuI.

The ligation and recleavage assay was replaced with LO test after validating experiments showed LO test ability to trace nuclease and phosphatase activities with sensitivity that is higher than L/R by a factor of 100.

No detectable degradation of a single-stranded and double-stranded labeled oligonucleotide was observed after incubation with 10 units of BcuI for 4 hours.

The B/W assay was replaced with LO test after validating experiments showed LO test ability to detect nuclease and phosphatase activities with sensitivity that equals to that of B/W test.

A minimum of 0.5units of BcuI is required for complete digestion of 1ug of Ad2 DNA in 16 hours at 37°C.

A minimum of 10 units of BcuI is required for complete digestion of 1ug of agarose-embedded Adenovirus-2 DNA in 16 hours.

Eco130I, NheI, XbaI, XmaJI

3

0

0

0

0

0

0

0

BcuI is not inactivated by incubation at 80°C for 20min.

(a) stop the digestion by adding 0.5M EDTA, pH 8.0, to achieve a 20mM final concentration. Mix thoroughly, add an electrophoresis loading dye and load onto gel.

(a) extract with phenol/chloroform, precipitate with ethanol or isopropanol, wash the pellet with 75% cold ethanol and air dry; (b) dissolve DNA in either nuclease-free water, TE buffer, or a buffer suitable for further applications; (c) check the DNA concentration in the solution.