338 Maunders
The above procedures refer to CLAP, since it is very difficult to
remove BAP after reaction. A newly available bacterial alkaline phos-
phatase isolated from an antarctic organism (28) can be heat killed.
After 30 min at 65°C, its activity is reduced to 0.01%.
4.3. Reaction Protocol
An example of a specimen reaction protocol is given in the following:
- Following restriction enzyme digestion, phenol/chloroform extraction,
and ethanol precipitation of the required DNA, dissolve the linearized
molecule in 10 mM Tris-HCl, pH 8.3.
- Take an aliquot containing 1 tag DNA.
- Add 10 I.tl-, of the following 10X reaction buffer:
- 100 mMTris-HCl, pH 8.3
- 10 mMMgC12
- 10 mM ZnC12
- Add water to 100 pL.
- Add 0.01 U of CIAP (diluted from stock in the specified buffer).
- Incubate at 37°C for 30 min.
- Add 5 ~L of 10% SDS and 5 ~L of 200 mM EDTA, pH 8.0. Mix well.
Heat to 65°C for 60 min.
- Extract with phenol/chloroform.
- Add 10 I~L of 3M sodium acetate, pH 7.0, and 300 ~ ethanol. Precipi-
tate the DNA at -20°C, and resuspend in the requisite buffer for subse-
quent manipulations (usually ligation).
- Summary
Alkaline phosphatase offers the molecular biologist another tool for
the manipulation of nucleic acids. Pretreatment with the enzyme
allows either the specific labeling of particular molecular species or
the correct synthesis of recombinant molecules. The wide specificity
of AP enables most substrates to be dephosphorylated, and by good
experimental design, this can be used to direct subsequent nonspecific
reactions, such as DNA ligation, to yield only the desired products.
The nature of the enzyme preparation and the variations in substrate
require particular reactions to be optimized, and the subsequent
removal of such a potent agent requires attention, but the simplicity
and effectiveness of the dephosphorylation reaction render it a very
widely used technique.
