Product description: Alkaline Phosphatase, TAB5 nonspecifically catalyzes the dephosphorylation of 5' and 3' ends of DNA and RNA phosphomonoesters. It also hydrolyses NTPs and dNTPs. It is completely inactivated by heating at 80℃ for 2 minutes.
Source: Recombinant E.coli
Concentration and Size: 2.5U/μL
Unit Definition: One unit is defined as the amount of enzyme that will dephosphorylate 1μg of pUC19 vector DNA cut with a restriction enzyme generating 5' recessed ends in 30 minutes at 37℃. Dephosphorylation is defined as ＞ 95% inhibition of recircularization in a self-ligation reaction and is measured by transformation into DH5α.
Storage Buffer: 10mM Tris-HCl , 1mM MgCl2, 0.01mM ZnCl2, 50%(v/v) glycerol, pH7.4, 25℃
10X Phosphatase Reaction Buffer: 500mM Bis-Tris-Propane-HCl , 10mM MgCl2, 1mM ZnCl2, pH6, 25℃
10X IVT Reaction Buffer without Mg2+ , Cat#ON-41, 400mM Tris PH7.9, 100mM DTT, 20mM Spermidine
High Yield T7 RNA Synthesis Kit, Cat#ON-040
T4 RNA ligase 1 (ssRNA ligase), Cat#ON-081
Ribonuclease Inhibitor, Human Placenta, Cat#ON-039
DNA as substance:
1. Combine the follows on ice,
|10X Phosphatase Reaction Buffer||2μL|
|Alkaline Phosphatase, TAB5||2.5~5 units|
|Nuclease-free H2O||To 20μL|
2. Incubate at 37℃, 30min
3. (optional) Heat inactivity enzyme by incubating at 80℃, 2min.
RNA as substance:
1. Heat Denature the RNA,
|Nuclease-free H2O||To 15μL|
2. Incubate at 65℃, 10~15min, transfer the tube immediately to ice.
3. On ice, combine the following reaction components in the order given
|15μL||Heat denatured RNA from step 1 (above)|
|2μL||10X Phosphatase Reaction Buffer|
|2.5~5 units||Alkaline Phosphatasae, TAB5|
|20 units||RNase inhibitor (Recommend)|
|20μL||Total reaction volume|
4. Incubate at 37℃, 30min
5. Stop the reaction using any one of the following methods:
a) immediate storage at –20℃ or –70℃.
b) Add of EDTA to a final concentration of > 2 mM.
c) Phenol/chloroform extract then precipitate using salt/alcohol.
Storage Conditions: -20℃
Quality Assurance: Free of endonuclease, exonuclease, nickase and RNase activities.
Physical Purity: ＞90% by SDS-PAGE.
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3. Ping Li, Jon Beckwith, et al. Proc. Natl. Acad. Sci. USA.85,7685-7689(1988).