Alkaline Phosphatase 337
pH 8.0-9.0 (or 100 mM Glycine/NaOH, pH 10.5) and 0.1-1.0 mM
ZnC12. In many cases, however, the presence of ZnCI 2 in the enzyme
storage buffer renders it unnecessary to add further zinc ions to the
reaction mixture. Further optional additions include 1 mM MgC12, 1 mM
Spermidine, and 0.1 mM EDTA (26).
The quantity of substrate and enzyme used ideally needs to be titrated
for each application (25), but is generally within the range of 1 U/l-100
pmol termini, with the larger enzyme:substrate ratios being employed
when the substrate is in the form of flush or recessed termini (26,38). The
substrate is usually present at a concentration of 1-50 pmol termini in a
reaction vol of 20-200 gL.
Optimum reaction temperatures and times also vary with substrate:- DNA carrying protruding phosphorylated termini can be dephosphory-
lated by reaction at 37°C for 30-60 min, with the optional addition of a
further aliquot of enzyme after 30 min (6,38,41). - DNA with flush ends will react under similar conditions (25), although
reaction at higher temperatures (e.g., 50°C) may be more efficient (26).
Two-step incubations of 15 min at 37°C followed by addition of more
enzyme and further incubation at 55°C for 45 min, or 15 min each at
37°C and 56°C followed by addition of more enzyme and a repetition
of the cycle give effective results (6). - DNA with recessed termini are best treated as for flush-ended DNA
(26), or by using BAP, higher temperatures can be employed, such as
incubations at 60°C for 60 min (25). - Dephosphorylation of RNA is also performed at higher temperatures,
such as 55°C for 30-60 min (41), possibly with an initial 15 min at
37°C (6).
After reaction, it is necessary to remove or inactivate the alkaline
phosphatase, since this will interfere with the efficiency of subsequent
ligation and transformation procedures. The reaction can be termi-
nated by adding EDTA to 10 mM, and then extracting with phenol. The
EDTA step may be improved by heating to 65°C for 60 min or 75°C
for 10 min prior to phenol extraction. Other methods of terminating the
reaction involve the digestion of ClAP by proteinase K (100 lag/mL in
5 mM EDTA, pH 8.0, 0.5% SDS) at 56°C for 30 min, or the chelation
of the zinc ions by heating to 65°C for 45-60 min in 50 mM EGTA, pH
8.0, or 10 mM nitrilo-triacetic acid (18). In all cases, a final extraction
with phenol or phenol/chloroform is recommended.