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Alkaline Phosphatase, Shewanella sp. SIB1, Recombinant (PAP)

Product#: FNK-DE110

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Alkaline Phosphatase, Shewanella sp. SIB1, Recombinant

Cat. No. FNK-DE110
Store at -20℃
Size 1000 Units
Concentration: 5unit/μl
Supplied reagent

10×Alkaline Phosphatase Reaction Buffer
Dilution Buffer

Storage Store at -20℃
Source

E.coli harboring the plasmid encoding the gene of alkaline phosphatase from a psychrotrophic bacterium Shewanella sp. SIB1 (PAP).

Unit Definition

One unit is defined as the amount required to hydrolyzed 1.0 μmole p-nitrophenyl phosphate per 1 minute in glycine-NaOH buffer at pH10.5 and 37℃.

Assay conditions

The reaction mixture (100μl) contains 50mM glycine-NaOH buffer, pH 10.5, 5 mM MgCl₂, 0.5 mM ZnCl₂, 10 0mM KCl, 5 mM p-nitrophanyl phosphate.

Storage Buffer

10mM Tris-HCl pH7.5
0.025mM ZnCl₂
0.25mM MgCl₂
50% glycerol

Description

Alkaline phosphatase from the psychrophilic strain Shewanella sp. SIB1 (PAP) has both merits of BAP and CIAP.

Alkaline phosphatase from the psychrophilic strain Shewanella sp. SIB1 (PAP) can easily be heat inactivated as SAP and CIAP.

On the other hand, BAP can not be easily inactivated even by phenol extraction.

Because activity of the enzyme at 60°C is about four times that at 37°C, the enzyme can easily removes phosphate from not only protruding 3'-end but also blunt end or recessed 5'-end at 60°C for 30min.

(On the other hand, SAP and CIAP were rapidly inactivated at 60°C.)

Contaminants

Dnase: When 0.5 μg of λ/Hind III digest was incubated with 10units of this enzyme in a 40 μl reaction mixture for 18 hours at 37℃, no degradation of the DNA fragment is observed on agarose gel electrophoresis.

Rnase: No RNase activity is observed by the use of RnaseAlert assay (Ambion). In this assay the reaction mixture containing the fluorescent-labeled RNA substrate was incubated with 10units of this enzyme for 1 hours at 37℃.

* Licensed Under Japan Patent NO. 2001-172653

Product Information

Composition of Supplied Reagent:

10×Alkaline Phosphatase Reaction Buffer ( Store at –20℃)

1. 5M	Tris-HCl, pH7.3
125mM	glycine
0. 5%	TritonX-100
0.25mM	ZnCl₂
2.5mM	MgCl₂
60mM	NiCl₂

Dilution Buffer ( 1×Reaction Buffer, Store at –20℃)

1. 5M	Tris-HCl, pH7.3
12.5mM	glycine
0. 05%	TritonX-100
0.025mM	ZnCl₂
0.25mM	MgCl₂
6mM	NiCl₂

Related Products

Gel Indicator (DM510)
DynaExpressDNA Ligation Kit ver.2 (DS110)

Advantage of PAP

Although BAP is hard to be inactivated, PAP is easily inactivated by heat.
As shown in figure below, PAP treatment produces a larger number of white colonies on plates than BAP treatment after transformation. Because active BAP survives after inactivation treatments, active BAP remove phosphate of insert DNA during the ligation reaction and result in the decrease the ligation efficiency. The experiment shows that PAP is easily inactivated before ligation but not BAP. In order to get sufficient amount of white colony after BAP treatment, insert DNA must be added at high insert: vector ratio into ligaton reaction to overcome surviving BAP or more than two times of phenol extraction must be carried out.

One μg of the EcoRI cleaved pBluescript SK (+) vector was dephosphorylated by 0.5 unit of BAP or 5 unit PAP at 37°C. After dephosphorylation, the reaction mixtures were treated as follow:

No-treatment: The reaction mixtures were directly used for ligation reaction.
Phenol: Equal volume of phenol was added to the reaction mixture and it was vortexed for 30 seconds. Then the mixture was extracted by ether, and precipitated by ethanol. The precipitate was dried up and dissolved by dH2O. It was used for ligation reaction.
Heat: The reaction mixtures were just heat at 95°C for 5 min for PAP or at 100°C for 5 min for BAP. These were directly used for ligation reaction.

After above treatments, EcoRI cleaved, dephosphorylated pBluescript SK (+) vector were ligated to a 1kb insert DNA fragment. Competent XL1-Blues were transformed with the ligation products. Number of white colony was shown in figure.