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        DEPHOSPHORYLATION OF LINEARIZED DNA

        互联网

        1260

        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℃.

         

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