Invitrogen™ Anza™ Alkaline Phosphatase is intended for use in the removal of 3' and 5' phosphate groups that remain after Anza™ restriction enzyme digestion, such as for the dephosphorylation of vectors prior to insert ligation and removal of 5′ phosphates prior to end-labeling. Since the 5′ phosphate group is required for DNA ligation, treatment of vectors with Anza Alkaline Phosphatase prevents self-ligation and re-circularization, resulting in decreased vector background when cloning.
Features of Anza Alkaline Phosphatase:
• Validated for use with all Anza restriction enzymes • Dephosphorylates in 15 minutes at 37°C • Heat inactivated after 5 minutes at 80°C • Fully active in Anza Buffer • Dephosphorylates all types of DNA ends (blunt, 5'- and 3'-overhangs)
Flexible protocol Some restriction enzymes prohibit or reduce the dephosphorylation process when digestion and dephosphorylation are performed simultaneously. Therefore, Anza Alkaline Phosphatase may be used in one of three ways depending upon the restriction enzyme used:
• One-Step Protocol: Simultaneous restriction enzyme digestion and dephosphorylation in Anza buffer • Heat Inactivation Two-Step Protocol: Digestion is carried out first, followed by heat inactivation (20 minutes at 80oC) then dephosphorylation • Column Purification Two-Step Protocol: Digestion is carried out first, followed by column purification then dephosphorylation
No need for enzyme removal Anza Alkaline Phosphatase is completely inactivated after 5 minutes at 80°C, so enzyme removal via phenol/chloroform extraction or column purification is not required prior to subsequent ligation steps. If the One-Step protocol was used, heat the reaction mixture for 20 minutes at 80oC to inactivate the restriction enzyme as well as the alkaline phosphatase. Heat inactivation of the alkaline phosphatase prevents residual phosphatase from dephosphorylating the DNA fragments added to the reaction mixture for ligation.