Background and overview[1]
Orthophosphatase, also known as orthophosphate monoester hydrolase, is an enzyme that can catalyze the hydrolysis of various phosphorus-containing compounds. According to their optimal pH characteristics for catalysis, it can be divided into acid phosphatase ( EC 3.1.32) and alkaline phosphatase (EC3.1.31). Phosphatase has a very important physiological function. It is widely present in various animals. It is an important detoxification system in the animal body. It also plays a role in the ossification process of the animal body and in the digestion, absorption and transportation of phosphide and other nutrients. plays an important role in both. Shrimp Alkaline Phosphatase (SAP) catalyzes the dephosphorylation reaction of 5′ and 3′ phosphate monoesters of DNA and RNA, and can also hydrolyze ribose and deoxyribonucleoside triphosphates (NTPs and dNTPs). This enzyme is widely used in molecular biology research, such as removing phosphate groups at the ends of DNA and RNA for subsequent cloning and probe end labeling. During cloning, after the vector is dephosphorylated by shrimp alkaline phosphatase, the 5’ end of the vector does not have a phosphate group, so it cannot be connected to its own 3’ end. Shrimp alkaline phosphatase can act on 5′ protruding ends, recessed ends and flat ends, and can also be used to degrade free dNTPs in PCR reactions for preparation of sequencing templates and SNP analysis. The shrimp alkaline phosphatase can be completely and irreversibly inactivated by incubation at 65°C for 5 minutes, so it does not need to be removed after ligation or end labeling. Based on the above characteristics, this enzyme is an excellent substitute for the traditional dephosphorylase-calf intestinal alkaline phosphatase.
Components
Apply[1]
Dephosphorylation of DNA and RNA;
Prevent self-ligation of cloning vectors;
Prepare 5´ end labeling template;
Remove dNTPs and pyrophosphates from PCR products.
Activity definition
1 unit refers to the amount of enzyme required to dephosphorylate 1 μg of pUC19 DNA digested by HindIII (producing 5’ overhanging ends) in 30 minutes at 25°C with aqueous amino resin. The dephosphorylation standard refers to the ability to inhibit >95% of DNA religation in self-ligation reactions.
Save
Can be stored at -20℃ for 3 years.
Precautions for use
(1) 1×SAP Buffer: 50 mM Bis-Tris HCl pH 6.0, 1mM MgCl2, 0.1 mM ZnCl2, incubate at 25°C.
(2) Thermal inactivation: Heating at 65℃ for 5 minutes, irreversible inactivation. This enzyme is commonly used for dephosphorylation of digested vectors due to its irreversible thermal inactivation properties. After the dephosphorylation reaction is completed, the product does not need to be purified and can be directly used in subsequent ligation reactions.
(3) Shrimp alkaline phosphorus Japanese mitritase can also be used to degrade free dNTPs in PCR reactions for preparation of sequencing templates and SNP analysis. It also requires no purification and can be used directly for downstream experiments.
Main references
[1] He Haiqi, Sun Feng. Research on the characteristics of acidic and alkaline phosphatase in Penaeus chinensis [D]. , 1992.
