Team:UNICAMP-Brazil/Protocols/CIAP Dephosphorylation


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CIAP Dephosphorylation

Protocol for Dephosphorylation of 5'-ends of DNA:

1. Dilute sufficient CIAP for immediate use in CIAP 1X Reaction Buffer to a final concentration of 0.01u/μl. Each picomole of DNA ends will require 0.01u CIAP. (1μg of 1,000bp DNA = 1.52pmol DNA = 3.03pmol of ends.)

2. Purify the DNA to be dephosphorylated by ethanol precipitation, and resuspend the pellet in 40μl of 10mM Tris-HCl (pH 8.0). Set up the following reaction:

- DNA (up to 10 pmol of 5´-ends) - up to 40μl

- CIAP 10X Reaction Buffer - 5μl

- Diluted CIAP (0.01u/μl) - up to 5μl

- MiliQ Water - to 50μl

3. Incubate at 37°C for 30 minutes.

4. Add another aliquot of diluted CIAP (equivalent to the amount used in Step 2), and continue incubation at 37°C for an additional 30 minutes.

5. Add 300μl of CIAP stop buffer. Phenol:chloroform extract and ethanol precipitate by adding 0.5 volume 7.5M ammonium acetate (pH 5.5) and 2 volumes of 100% ethanol to the final aqueous phase.

Protocol according to Promega