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 Å 1.4pmol 5'-ends

3. Incubate 30' @ 37℃.

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 @ Å100ng/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' @ 37℃.

4. Add another aliquot of CIP and incubate an additional 45' @ 55℃.

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'@ 37℃, followed by 30' @ 55℃.