====== TEV preparation - Séraphin Lab ======

**j-1** 
  * Transform BL21/LysS strain on KanR with optimized TEV plasmid (stock pBS2440).

**j** 
  * Inoculate 25 ml medium A((LB, Cm 25 μg/ml, Kan 10 μg/ml)) with one colony at the end of the afternoon.
  * Grow at 37°C.

**j+1** 
  * Inoculate 1 L medium A with the 25 ml preculture. 
  * Grow at 30°C until OD600 reach 0.9.
  * Add IPTG to the medium up to 0.1 mM. 
  * Grow 6 hours at 23 °C.
  * Pellet cells 20 min at 4,000 rpm  in GS3. 
  * Wash with PBS 1X.
  * Freeze pellet in liquid N2 and store at -80°C.

**j+2** 
  * Resuspend pellet in Buffer 1((20 mM Tris-HCl pH 8.0, 10 mM imidazole, 200 mM Nacl, 2 mM β-ME, 0.2% NP40)) 
  * Adjust to 20 ml with Buffer 1.
  * Pass through French-Press 2 times.
  * Spin down with ss34 12,000 rpm for 30 min.
  * Put the supernatant with 1 ml slurry of NiNTA beads (previously washed with buffer 1) in a Falcon tube. 
  * Rotate 1h at 4°C. 
  * Transfer in an Econo-column (Biorad).
  * Wash with 15ml Buffer 1.
  * Wash with 5 ml Buffer 2((20 mM Tris-HCl pH 8.0, 10 mM imidazole, 1 M NaCl, 2 mM β-ME, 0.2% NP40))
  * wash with 10 ml Buffer 1.
  * Elute with 4x0.5 ml Buffer 3((20 mM Tris-HCl pH 8.0, 200 mM imidazole, 200 mM NaCl, 2 mM β-ME, 0.2% NP40)) in 0.5 ml glycerol tube (50% glycerol final).
  * store at -80°C.

You may perform an additional chromatography step fort higher purity.