DNA Reaction - Phosphatasing

Jeff Lawrence

1. Following restriction endonuclease digestion, inactivate enzyme and dislodge it from DNA ends by incubating at 65° for 5 min.

2. Dilute Calf Intestine Alkaline Phosphatase 1:100 in 50 mM Tris to a concentration of 0.01 units/ mL

3. Add 1 µL of Dilute Alkaline Phosphatase per pmol of 5' protruding ends. Add 10 µL per pmol for blunt or 5' recessed ends.

4. Incubate at 37° for 30 min for protruding ends. Incubate at 50° for 60 min for blunt or 5' recessed ends.

5. Add 1 M EGTA to a final concentration of 50 mM.

6. Incubate at 68° for 10 min.

7. Extract and precipitate, adding 1% Polyacrylamide carrier if needed.

Last Update: Thursday June 19 2014

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Eric Kofoid eckofoid at ucdavis.edu