Krishzyme 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 labelling 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.
Unit Definition
One unit is defined as the amount of enzyme that will dephosphorylate 1 g of pUC19 vector DNA cut with Hind III (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.
Molecular Weight
The molecular weight of Alkaline Phosphatase is 35 kDa.
Application:
– Dephosphorylation of DNA and RNA
– Prevention of recircularization of cloning vectors
– Removal of dNTPs and pyrophosphate from PCR reactions
– Preparation of templates for 5 end labelling