Using Shrimp Alkaline Phosphatase (SAP)

1. Clean up the DNA using this abbreviated protocol. You do NOT want to use SAP in the restriction digestion buffer. Resuspend DNA in water. Perform this procedure in a 500 μL microfuge tube.

2. Add appropriate volume of 10X SAP buffer to your DNA. Final volume should be 30 μL.

3. Add appropriate amount of SAP enzyme. Keep enzyme cold at all times. Incubate 1 unit of SAP for every microgram of cut DNA.

4. Incubate SAP with DNA for 15 minutes at 37° C.

5. Inactive SAP by incubating the DNA for 15 minutes at 65° C.

6. Either go directly to ligation, or gel purification.

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