DEPHOSPHORYLATIONOFLINEARIZEDDNA

DEPHOSPHORYLATION OF LINEARIZED DNA
ALKALINE PHOSPATASE(CIP) DIGESTION

Digestion of protruding 5'-ends
1. Precipitate digested DNA and resuspend in a 100ml of 1X CIP Buffer.
2. Add 1U CIP for 100pmol 5'-ends.
2g linearized 5kb plasmid ? 1.4pmol 5'-ends
3. Incubate 30' @ 37oC.
4. Add 1ml 0.5M EDTA (to 5mM).
5. Phenol-Sevag extract to remove all the enzyme and NaOAc/EtOH ppt.
6. Resuspend in ddH2O or TE-4 @ ?100ng/l.

Calf Intestinal Alkaline Phosphatase
Boehringer Mannhein #713 023

0.1M Tris-HCl, pH 8.5
10mM ZnCl2
10mM MgCl2

Digestion of blunt or recessed termini
1. Precipitate digest DNA and resuspend in 100ml 1X CIP Buffer.
2. Add 1U CIP per 2 pmol -ends.
3. Incubate 15' @ 37oC.
4. Add another aliquot of CIP and incubate an additional 45' @ 55oC.
5. Stop rxn by adding 1ml 0.5M EDTA, phenol-Sevag extract and EtOH ppt.

Dephosphorylation of RNA
To remove 5' phosphates from RNA, use 0.01U CIP/pmol 5' termini. Incubate 15'@ 37oC, followed by 30' @ 55oC.