Materials:

1.     Lysis Buffer : 20 mM Tris-HCl (pH 7.5), 150 mM NaCl and 0.2% Triton X-100. (Do not add EDTA since it inactivates CIP.)

2.     EDTA-free Protease Inhibitor Cocktail : #11873580001, Roche

3.     CIP Buffer: 50 mM Tris-HCl (pH 7.9), 100 mM NaCl, 10 mM MgCl₂, and 1 mM Dithiothreitol (DTT)

4.     CIP (calf intestinal phosphatase): M0290, New England Biolabs

Reference:

Robison, J.G., Elliott, J., Dixon, K., and Oakley, G.G. 2004. Replication protein A and the Mre11.Rad50.Nbs1 complex co-localize and interact at sites of stalled replication forks. The Journal of biological chemistry 279: 34802-34810.