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.
