Description
Alkaline Phosphatase is derived from a recombinant E. coli strain that carries the TAB5 gene. The enzyme catalyzes the dephosphorylation of 5´ and 3´ ends of DNA and RNA phosphomonoesters. Also, it hydrolyses ribose, as well as deoxyribonucleoside triphosphates (NTPs and dNTPs). TAB5 Alkaline Phosphatase acts on 5´ protruding, 5´ recessed and blunt ends. The Phosphatase can be used in many molecular biology applications, such as cloning or probe end labeling to remove the phosphorylated ends of DNA or RNA. In cloning experiments, dephosphorylation prevents the linearized plasmid DNA from self-ligation. It can also degrade unincorporated dNTPs in PCR reactions to prepare a template for DNA sequencing. The enzyme is completely and irreversibly inactivated by heating at 70°C for 5 minutes, thereby making removal of the phosphatase prior to ligation or end labeling unnecessary.
Enzyme Commission Number
EC 3.1.3.1
Unit Definition
One unit is defined as the amount of enzyme that will dephosphorylate 1 µg of pUC19 vector DNA cut with HindIII (5´ protruding ends), HincII (blunts ends) or PstI (5´ recessed ends) in 30 minutes at 37°C. Dephosphorylation is defined as > 95% inhibition of recirculation in a self-ligation reaction and is measured by transformation into E.coli.
Storage
at -20 °C (Avoid repeated freeze-thaw cycles)
Buffer
10 mM Tris-HCl (pH 7.4,25°C), 1 mM MgCl2, 0.01 mM ZnCl2, 50% glycerol.