13. Alkaline Phosphatase (Thermosensitive), 1000 U, $100

Product Information

Description/Introduction

Alkaline phosphotase (thermosensive) produced by our company is an alkaline phosphatase purified by recombinant expression of heat labile Escherichia coli from Antarctic microorganism (TAB5), which can non specifically catalyze the removal of 5 ‘or 3’ terminal phosphate groups of DNA, RNA, dNTP and rNTP. Alkaline phosphotase is widely used in molecular biology, such as removing phosphorylated ends of DNA and RNA for subsequent cloning or end labeling of probes; In cloning, dephosphorylation can prevent self ligation of linearized plasmid DNA; It can also be used to degrade unbound dNTP in PCR reactions for subsequent sequencing or SNP analysis.

Features: rapid dephosphorylation; Thermal instability, incubation at 80 ℃ for 2 min can completely inactivate.

Source: E. coli strain carrying tab5 alkaline phosphatase gene; Its monomer molecular weight is about 35 kDa;

Enzyme activity definition: the amount of enzyme required for dephosphorylation of 1 µ g linearized pUC19 vector was defined as one enzyme activity unit after incubation at 37 ° C for 30 min. Dephosphorylation was defined as the inhibition rate of carrier autoligation reaction greater than 95% (measured by E. coli transformation).

Inactivation or inhibition: heating at 80 ℃ for 2 min can fully inactivate alkaline phosphorase. Metal ion chelating agents such as EDTA can inhibit alkaline phosphorase.

Purity and concentration: SDS-PAGE detection purity ≥ 95%, 5 u/ μ L.

Enzyme storage buffer: 10 mm Tris HCl, 1 mM MgCl2, 0.01 mm ZnCl2, 50% glycorol, pH 7.4.

ten × ALPReaction Buffer：500 mM Bis-Tris-HCl，10 mM MgCl2，1 mM ZnCl2，pH 6.0.

Storage and Handling Conditions

Transport with wet ice. Store at -20°C, valid for 12 months.

Component

Assay Protocol / Procedures

1. Dephosphorylation reaction reference system:

2. After being prepared according to the above system, mix it gently, and then centrifuge the liquid to the bottom of the tube.Incubate at 37℃ for 30 minutes, then add 0.5 μL 25 mM EDTA to the reaction system to stop the reaction;

3. Incubate at 37 ℃ for 15-30 min. For better dephosphorylation effect, the incubation time can be extended to 60 min.

4. Alkaline phosphorase was inactivated by heating at 80 ℃ for 2 min.

5. According to the needs of subsequent experiments, appropriate DNA purification kit or DNA gel recovery kit can be used for gel cutting recovery and purification after gel electrophoresis, or phenol chloroform extraction, ethanol precipitation and other methods can be used to purify the digested and dephosphorylated DNA or RNA.

Note:

1. Alkaline phosphatase is the same as other alkaline phosphatases, and its enzymatic activity requires the presence of Zn2 + and Mg2 +. ALP reaction buffer can provide enough Zn2 + and Mg2 + to ensure the enzyme activity.

2. Alkaline phosphatase is inhibited in the presence of metal ion chelating agents such as EDTA, inorganic phosphates and phosphate analogues.

3. The activity of alkaline phosphotase decreases in the presence of reducing agents such as DTT and mercaptoethanol.

4. When using the product, the enzyme should be stored in an ice box or on an ice bath. After use, it should be stored at – 20 ℃.

5. For your safety and health, please wear lab coat and disposable gloves to operate.

For Research Use Only!

Ver. No.: V1.0-202208