Thermosensitive Alkaline Phosphatase
Rapid dephosphorylation; heat-labile, completely inactivated by 2 min at 80°C.
Gato. No. :
Y101631-1000UMarca :
YanbiotechOrigen del producto :
ChinaVolumen :
1000UProduct Information：
| Product Description: | Yanbiotech Alkaline Phosphatase (Thermosensitive) catalyzes the release of 5'- and 3'- phosphate groups from DNA、RNA、dNTP and rNTP. This enzyme also removes phosphate groups from proteins. |
| Applications: |
Dephosphorylation of cloning vector DNA to prevent recircularization during ligation. PCR product clean-up: nucleotide degradation prior to sequencing of PCR product. Dephosphorylation of nucleic acid 5’-termini prior to labeling with T4 Polynucleotide Kinase. |
| Source: | E.coli with a cloned bacterial AP gene form AntarcticBacteriumTAB5. |
| Purity: | ≥95% by SDS-PAGE |
| Molecular Weight | ~35 kDa |
| Concentration: | 5 U/μL |
| Definition of Activity Unit: | One unit is the amount of enzyme required to catalyze the dephosphorylation of 1 µg of linearized pUC19 DNA 5' end in 10 minutes at 37℃. Dephosphorylation is defined as greater than 95% inhibition of religation of linearized plasmid DNA (as determined by E. coli transformation). |
| 10× ALP Reaction Buffer: | 500 mM Bis-Tris-HCl，10 mM MgCl2，1 mM ZnCl2，pH 6.0. |
| Inactivation or inhibition |
Inhibitors: metal chelators. · Inactivated by heating at 80°C for 2 minutes. |
| Storage Conditions: | Store at –20°C up to 12 months. |
Product Contents：
| Component Number | Component | Y101631-1000U |
| Y101631-1000U-1 | Alkaline Phosphatase (Thermosensitive) | 200 μL |
| Y101631-1000U-2 | 10×ALP Reaction Buffer | 1 mL |
Instructions for Use:
This protocol is suitable for removal of 3’and 5’-phosphate groups from DNA and RNA.
Thaw frozen reagents, mix and centrifuge briefly.
Keep enzymes on ice.
Keep the 10×ALP Reaction Buffer at room temperature.
1. Prepare the following reaction mixture containing:
| Component | Volume |
| DNA or RNA sample | 1-5 μg |
| 10×ALP Reaction Buffer | 2 μL |
| Alkaline Phosphatase (Thermosensitive | 1 μL |
| Nuclease-Free Water | Add to 20 μL |
| Total volume | 20 μL |
2. Mix thoroughly, spin briefly and incubate at 37°C for 15-30 mininutes. The optimal incubation time and the enzyme concentration must be determined experimentally for each substrate.
3. Stop reaction by heating for 2 minutes at 80°C.
4. According to the needs of subsequent experiments, the above dephosphorylated DNA or RNA can be purified in a variety of ways (such as DNA purification kit, phenolic chloroform extraction and ethanol precipitation method).
Notice:
1. Alkaline Phosphatase as are most alkaline phosphatases, is a Zn2+and Mg2+-dependent enzyme. The ALP Reaction Buffer provides enough Zn2+and Mg2+ to guarantee enzyme activity.
2. Alkaline Phosphatase is inhibited by metal chelators (e.g. EDTA), inorganic phosphate and phosphate analogs.
3. The Alkaline Phosphatase activity is decreased in the presence of reducing agents (DTT, β-ME).
4. The Alkaline Phosphatase should be shipped on ice when handling, and should be stored at -20 ℃ immediately after use.
5. For your safety and health, please wear safety glasses, gloves, or protective clothing.
6. For Research Use Only
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