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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
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