Manual

Product Number: S120059

Shipping and Storage

Ship with wet ice; Store at -20℃ valid for 12 months.

Component

Description

Alkaline Phosphatase (Thermosensitive) is a thermolabile enzyme derived from Antarctic microorganisms (strain TAB5), which has been recombinantly expressed and purified using Escherichia coli. This enzyme catalyzes the nonspecific removal of 5' or 3' terminal phosphate groups from DNA, RNA, dNTPs, and rNTPs. It is widely applied in molecular biology, including the dephosphorylation of DNA and RNA termini to facilitate probe cloning or end labeling. In molecular cloning, dephosphorylation of linearized plasmid DNA prevents self-ligation. Additionally, the enzyme can be employed to degrade excess unincorporated dNTPs in PCR products prior to sequencing or SNP analysis.

Application

1. Dephosphorylation of cloning vector DNA to prevent recircularization during ligation.
2. Simultaneous digestion and dephosphorylation of vector DNA.
3. PCR product clean-up: nucleotide degradation prior to sequencing of PCR product.
4. Dephosphorylation of nucleic acid 5’-termini prior to labeling with T4 Polynucleotide Kinase.
5. Other applications where dephosphorylation of DNA and RNA substrates is necessary.

Source

 E.coli cells with TAB5 alkaline phosphatase gene.

Definition of Activity Unit

One unit is the amount of the enzyme required to dephosphorylate 5'-termini of 1µg of linearized pUC19 DNA in 30 min at 37°C in ALPReaction Buffer. Enzyme activity is assayed in the following 500uL mixture: 100mM Tris-HCl (pH 7.2), 2mM MgCl2 and 2mM PPi, pH 8.0; Reaction at 25℃ for 10 min.

Inhibition and Inactivation

1. Inhibitors: metal chelators.
2. Inactivated by heating at 80°C for 2 min

Purity and concentration

Purity ≥95% by SDS-PAGE; 5 U/uL.

Buffer

1. Storage (Dilution) Buffer: 10mM Tris HCl, 1mM MgCl₂, 0.0 mM ZnCl₂, 50% glycorol, pH 7.4.
2. 10×ALP Reaction Buffer：500mM Bis-Tris-HCl, 10mM MgCl₂, 1mM ZnCl₂, pH 6.0.

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