This is the procedure we tried for phosphatization:
Step 1: Add 1/10 volume of 10x Antarctic Phosphatase Reaction buffer to 1 ug of DNA cut w/ any restriction endonuclease in any buffer.
Step 2: Add 1 ul (~5 units) of (A-Pase)/mix.
Step 3: Incubate for 15 min. @ 37 C for 5' extens. or blunt-ends; 60 min. for 3' extens.
Step 4: Heat inactivate for 5 min. @ 65 C (or as required to inactivate the restriction enzyme).
Step 5: Proceed w/ ligation.