Nuclei preparation buffer II

0.3 M sucrose

60 mM KCl

15 mM NaCl

5 mM MgCl₂

0.1 mM EGTA

15 mM Tris-Cl (pH 7.5)

0.5 mM DTT

0.1 mM PMSF

3.6 μg/ml aprotinin

0.4% (v/v) IGEPAL CA-630

Protease inhibitors, and in particular PMSF, are rather unstable in aqueous solutions. Therefore, PMSF, as well as DTT and aprotinin, should be added as late as possible, just before use.

For many tissues (liver, kidney, placenta), a final concentration of 0.2% of the nonionic detergent IGEPAL CA-630 will be enough to lyse the cellular membranes during the 10-minute incubation. However, we recommend testing 0.4% IGEPAL CA-630 for other tissues. For example, this higher concentration of detergent slightly improves the yield of nuclei from brain and muscle tissues.