Method

1. Digest a reasonable quantity of closed circular plasmid DNA (10 Mg) with a two- to threefold excess of the desired restriction enzyme for 1 hour.

2. Remove an aliquot (0.1 Mg), and analyze the extent of digestion by electrophoresis through a 0.7% agarose gel containing ethidium bromide, using undigested plasmid DNA as a marker. If digestion is not complete, add more restriction enzyme and continue the incubation.

3. When digestion is complete, extract the sample once with phenol:chloroform and recover the DNA by standard precipitation with ethanol. Store the ethanolic solution on ice for 15 minutes.

4. Recover the DNA by centrifugation at maximum speed for 10 minutes at 4°C in a microfuge, and dissolve the DNA in 110 Ml of 10 mM Tris-Cl (pH 8.3).

5. To the remaining 90 Ml of the linearized plasmid DNA, add 10 Ml of 10x CIP or 10x SAP buffer and the appropriate amount of calf intestinal phosphatase (CIP) or shrimp alkaline phosphatase (SAP) and incubate as described in the table below.

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