Alkaline Phosphatase recombinant, Highly Active Protocol

Product No. 03359123001

Protocol

Buffer for dephosphorylation using Alkaline Phosphatase

Alkaline Phospatase, special quality for molecular biology is active in all five SuRE/Cut Buffers (Incubation Buffers for restriction enzymes), but to varying extents.
AP activity varies in the following order regarding the type of SuRE/Cut Buffer:
A >= B > L = M = H.
Salt is not the critical parameter, but the pH:
Buffer B = pH 8.0, Buffer A = pH 7.9, Buffers L, M, H = pH 7.5.
For best results, adjust the pH to 8.0 after restriction enzyme digestion with a tenth volume of 500 mM Tris/pH 8.0 before adding Alkaline Phosphatase.

Inactivation of Alkaline Phosphatase

Alkaline Phosphatase can generally be inactivated by heat: Heat the reaction for 10 min at +65 °C, followed by at least one extraction with phenol/chloroform/isoamylalcohol (50 : 48 : 2).
AP inactivation using heat depends upon the protein concentration and the divalent ion concentration (Mg2+, Zn2+) of the preparation. For inactivation of AP, Molecular Biology Grade following a dephosphorylation reaction, heat the reaction 30 min at +65 °C in the presence of 50 mM EGTA, not EDTA;, as EGTA is needed to chelate divalent cations. Then extract the solution with phenol and precipitate the nucleic acids with ethanol.
The amount of EGTA needed depends upon the Mg2+ concentration of the reaction mix: If the reaction mix contains no Mg2+ use 5 mM EGTA (final conc.); if the reaction mix contains 1 mM Mg2+ use 10 mM EGTA.
Phenol extraction should always be included in the inactivation procedure when the AP concentration is higher than 0.3 U/10 μl, to insure that AP does not interfere with subsequent steps. When time or amount of nucleic acid preclude ethanol precipitation, a longer incubation (up to 45 min) at+ 65 °C in the presence of EGTA is usually be sufficient to completely inactivate AP. During the heating step, centrifuge the reaction mix briefly (for few seconds) every 10 min to spin down liquid which has condensed on the tube walls.

Materials

     

SURE/CUT is a trademark of Roche

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