Team:UNICAMP-Brazil/Protocols/SAP Dephosphorylation

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SAP Dephosphorylation
Protocol for Dephosphorylation of 5'-ends of DNA:

1. Resuspend 1 pmol of DNA ends (about 1 μg of a 3 kb plasmid) in nuclease-free water.

2. Prepare reaction mix in a 20 μl volume according to the following:

- DNA - 1 μl

- 10X SAP Reaction Buffer - 2 μl

- Water, Nuclease-Free - up to 19 μl

- Shrimp Alkaline Phosphatase (1 unit/μl) - 1 μl

3. Incubate at 37°C for 30-60 min.

4. Stop reaction by heating at 65°C for 15 min. This completely inactivates SAP.

Protocol according to USB www.usbweb.com