What are the recommended conditions for proteinase K treatment when isolating RNA or DNA samples?
Concentration: Generally proteinase K is used in the concentration range of 50 to 500 µg/mL at 65 degrees C in the presence of SDS (0.5-1%).
Temperature optimum: 65 degrees C; 12X more active at 65 degrees C than at 25 degrees C.
pH: Proteinase K is stable over a wide pH range (4.0 to 12.5), with optimal activity at pH 6.5 to 9.5. It is most stable at pH 8.
Inactivation: Heat inactivate Proteinase K at 80 degrees C for 15 min. Phenol extraction is the recommended method to ensure complete inactivation.
Inhibited by: PMSF (0.1 to 1.0 mM of PMSF is usually sufficient for inhibition of Proteinase K)
Not inhibited by: Proteinase K is not inactivated by metal ions, chelating agents (e.g., EDTA), sulfhydryl reagents or by trypsin or chymotrypsin inhibitors. Activity can be stimulated by addition of denaturing agents (SDS and urea).
Temperature optimum: 65 degrees C; 12X more active at 65 degrees C than at 25 degrees C.
pH: Proteinase K is stable over a wide pH range (4.0 to 12.5), with optimal activity at pH 6.5 to 9.5. It is most stable at pH 8.
Inactivation: Heat inactivate Proteinase K at 80 degrees C for 15 min. Phenol extraction is the recommended method to ensure complete inactivation.
Inhibited by: PMSF (0.1 to 1.0 mM of PMSF is usually sufficient for inhibition of Proteinase K)
Not inhibited by: Proteinase K is not inactivated by metal ions, chelating agents (e.g., EDTA), sulfhydryl reagents or by trypsin or chymotrypsin inhibitors. Activity can be stimulated by addition of denaturing agents (SDS and urea).