Heat Inactivated Alkaline Phosphatase is a phosphomonoesterase that hydrolyzes 3’ and 5’ phosphates from DNA and RNA. It is suitable for removing 5’ phosphates prior to end-labeling and for dephosphorylating vectors prior to insert ligation. It can also be used to dephosphorylate proteins.
Features of Heat Inactivated Alkaline Phosphatase:
- Removes 5′ phosphate groups from DNA, RNA, and protein and dNTPs from PCR reactions
- Dephosphorylates in 15 minutes at 37 °C
- Heat inactivated in 5 minutes at 65 °C
- Compatible with Universal Restriction Enzyme buffers L, M, H, K, and T
Reduce Vector Background, Clean Up PCR Reactions, Dephosphorylate Proteins
Since the 5′ phosphate group is required for DNA ligation, treatment with Heat Inactivated Alkaline Phosphatase prevents vector self-ligation and recircularization, resulting in decreased vector background when cloning. In addition, Heat Inactivated Alkaline Phosphatase can be used to remove dNTPs and pyrophosphate from PCR reactions and to dephosphorylate proteins.
Easily Inactivated So Removal Is Not Necessary
Heat Inactivated Alkaline Phosphatase is completely inactivated in 20 minutes at 65 °C, so no phenol/chloroform extraction or column purification is required to remove the enzyme prior to ligation. This means DNA fragments added to the reaction mix for ligation are not treated with residual phosphatase and will be ligated to the dephosphorylated vector DNA.
Heat Inactivated Alkaline Phosphatase Buffers:
- Heat Inactivated Alkaline Phosphatase Storage Buffer: 10 mM Tris-HCl (pH 7.5); 1 mM MgCl2, 0.1 mM ZnCl2, 1 mM DTT, 50% (v/v) glycerol.
- 10X Reaction Buffer: 100 mM Tris-HCl (pH 7.5); 100 mM MgCl2, 10 mM DTT.