Seydoux lab Cas9 preparation
Transform DE3 GOLD ( Agilent , # 230132 ) cells with nm2973 plasmid ( Fu et al 2014 ) and plate on LB + 50μg / mL Carbenicillin .
Inoculate 25mL LB + 50 μg / mL Carbenicillin with bacteria from the fresh transformation and incubate at 37 °C overnight .
Transfer 5mL of overnight culture to 1L LB + 0.1 % glucose + 50 μg / mL Carbenicillin .
Grow at 25 °C to OD600 = ~ 0.5 .
Shift culture to 18 °C for 15‐25 minutes .
Add IPTG to 0.2 mM and incubate overnight .
Pellet culture and obtain wet weight .
Resuspend at ~ 6 mL / g cells with Buffer A + protease inhibitor ( Roche , # 11836170001 ) + 1mM PMSF .
Sonicate 6 x 45s ( setting 3 at 30 % , 1 second pulse‐2 second pause ) with 1 minute cooling inbetween .
Spin lysate 30 minutes at 16000xg and transfer supernatant to a fresh tube .
Equilibrate a 5mL Ni‐agarose ( Qiagen , # 30410 ) with column with Buffer A ( at least 25mL ) .
Batch bind clarified lysate with Ni‐agarose 45 minutes at 4 °C .
Wash Ni‐agarose column with 100mL of Buffer B .
Elute protein with Buffer C .
Determine fractions that have Cas9 protein using Bradford assay or by running a small amount on SDS‐PAGE gel .
Pool fractions .
Equilibrate a 5mL Q Sepharose ( Sigma , # Q1126 ) column with 1M KCl ( 25mL , this charges the column ) .
Equilibrate Q Sepharose column with Buffer C ( 25mL ) .
Flow eluent ( from step 17 ) over Q Sepharose column .
Collect flow‐through and dialyze into 1L Buffer D for 5 hours at 4 °C .
Transfer into 1L Buffer D and dialyze overnight .
Concentrate protein to ~ 10 mg / mL using a 100K centrifugal filter ( Milipore , UFC910024 ) .
Aliquot and flash‐freeze in liquid nitrogen .
Store aliquots at ‐80°C .
